**5. DAC® hydrogel** *in vitro* **activity**

### **5.1. Cell compatibility assay**

Preparation of the hydrogel was performed according to a patented procedure [49]. In particular, HA-g-PLA copolymer was dispersed in an appropriate volume of twice distilled water, and the mixture was stirred vigorously at the vortex to obtain a gelatinous and transparent

The synthesis of HA-g-PLA copolymer was performed as previously reported [50, 51] (**Figure 3**). Briefly, a low weight–average molecular weight HA (HALMW) was made soluble in organic solvents by transformation to its tetrabutylammonium (TBA) salt. The synthesis of the N-hydroxysuccinimide (NHS) derivative of PLA (i.e., PLA-NHS) was performed as reported elsewhere [52]. In particular, 2.4 g of PLA was dissolved in 30 ml of anhydrous dichloromethane with an excess of DCC and NHS for 24 h at room temperature, and then the solution was precipitated in ethanol and the recovered solid was dried under vacuum. 1H NMR of PLA-NHS

)─OH; d, 3H, ─O─CO─CH(CH3

)─O─),

hydrogel with a polymer concentration between 3% (w/v) and 10% (w/v).

) showed: *δ* 1.5 and *δ* 1.6 (d, 3H, ─O─CO─CH(CH3

**Figure 3.** Principal steps in the synthesis of HA-g-PLA copolymer.

(CDCl3

184 Hydrogels

*In vitro* cell compatibility of DAC® HA-g-PLA hydrogel (polymer concentration 6%, w/v) was evaluated using human dermal fibroblasts. The viability of cells cultured in direct or indirect contact with HA-g-PLA hydrogel was comparable with that of the control well, showing that the hydrogel does not release in the culture medium substances that interfere with cell viability and they do not cause a decrease in the cell viability after direct contact with them [24]. Further *in vitro* and *in vivo* biocompatibility studies were performed on the DAC® hydrogel and on the DAC® kit, in accordance to ISO standards, all showing no cytotoxicity, genotoxicity, sensitization, irritation or intracutaneous reactivity, systemic toxicity (acute), subchronic toxicity or interference with bone or periimplant tissues.

Furthermore, as degradation of DAC® HA-g-PLA hydrogel occurs via deesterification of hyaluronic acid and polylactic acid, it gives raise exclusively to the starting macromolecules, whose degradation pathways in the human body are widely known and whose use as implantable class III medical devices is largely accepted and tested safe.

#### **5.2. Antiadhesive and antibiofilm activity**

Both the ability of the DAC® HA-g-PLA hydrogel to reduce bacterial adhesion and biofilm formation were extensively studied *in vitro*.

Reductions of adhered bacteria on sterile titanium discs, coated with DAC® hydrogel, equal to 86.8, 80.4, 74.6 and 66.7% vs. untreated discs were observed after 15, 30, 60 and 120 min of incubation, respectively [37]. In another experiment, the ability to dislodge previously adhered bacteria was investigated. Once again, the results showed that DAC® hydrogel treatment of discs reduced the amount of adhered bacteria in respect to control discs after 15, 30, 60 and 120 min by 84.0, 72.8, 72.3 and 64.3%, respectively [37].

process that schematically includes the bacterial adhesion to a substrate, the subsequent immediate release of signals from adherent bacteria that triggers biofilm production and, finally, the biofilm construction and progressive consolidation. Acting mainly as a physical and antiadhesive barrier, DAC® hydrogel may reduce or prevent the first phase of the process, provided that the number of living bacteria is not too high and that they are not able to overcome or hydrolyze the hydrogel [54]; moreover, for an effective prevention of bacterial colonization of the coated implant, it is necessary that, while bacteria are in the more vulnerable planktonic state, they are completely removed or killed by the host's immune system and/or by the local chemicophysical environment. This is why, even in the presence of DAC® coating protection, systemic antibiotic prophylaxis is still to be considered necessary. In fact, if not eliminated, the remaining floating microorganisms may successfully colonize the implant once the protective coating has been hydrolyzed (a phenomenon that is expected to happen normally within 3 days from application for the DAC hydrogel) or after the implanted biomaterial has been covered by host's proteins (fibrin, fibronectin, etc.), which may also work as suitable for bacterial adhesion. In this scenario, the possibility to add also an antibacterial drug to the coating may further contribute to reduce the planktonic microorganisms, enhanc-

Hyaluronic-Based Antibacterial Hydrogel Coating for Implantable Biomaterials in Orthopedics…

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187

To test the hydrogel ability to entrap antibacterial drugs, vancomycin and tobramycin had been originally chosen as examples of antibiotic molecules [24]. Both these antibiotics have been added to the hydrogel just before its use, a solution that offers several advantages. First of all, this allows to add the antibiotic when it is needed, thus avoiding the problems of shelflife and any long-term compatibility with the hydrogel; secondly, in this way, it is possible to choose the optimal antibiotic in the specific case, taking into account the patient's specificity (e.g., known intolerances to specific antibacterial agents) or of the specific intervention; finally, the dosing of the antibacterial agent on a case-by-case basis can be decided by the clinician. The results of the *in vitro* study clearly showed how the investigated antibacterial hydrogel coating, applied on a titanium disc, at a concentration in polymer in the range 2–8% (w/v) and a concentration in drug equal to 1 or 2% (w/v) is able to release vancomycin or tobramycin, or of their association, for up to 72 h, with an amount of drug released that is hundred or thousand times higher than the minimum inhibitory concentration (MIC), in a

Similar results were founded by testing several other antibacterial compounds or their combinations, including vancomycin, teicoplanin, rifampicin, daptomycin, tigecycline, cefazolin, gentamicin, tobramycin, amikacin, meropenem, levofloxacin, etc. (cf. **Figure 5**), at concentra-

In summary, all examples reported above show that DAC® hydrogel is potentially able to entrap and release suitable quantities of antibacterial agents just after the implant of the coated prosthesis. The high initial burst effect of the released drug may ensure the most efficient action at the time that is critical for the destiny of the biomaterial. Moreover, during the entire drug release period, the antibiotic concentration released by the hydrogel remains greater than MIC, thus further ensuring effectiveness of the drug released in proximity to the

ing the overall protection offered by the DAC hydrogel (**Figure 4**).

time- and dose-dependent manner.

tions ranging from 2 to 10% [48].

prosthesis.

Concerning more specifically the antibiofilm activity, DAC® hydrogel showed similar or superior *in vitro* activity, compared to various antibacterials and a synergistic activity when used in combination [48]. In one experimental setting, *S. epidermidis* and *S. aureus* were grown on chrome-cobalt devices in 6-wells polystyrene plates containing TSB for 24 h at 37°C. The plates were incubated at 37°C in ambient air, until a visible biofilm was obtained. Gentamycin and vancomycin were tested at a final concentration of 20 mg/mL. Similarly, when mixed with the hydrogel, 60 mg of gel powder was reconstituted with 1 mL of water for injections containing gentamicin or vancomycin at 20 mg/mL concentration. The amount of biofilm at each time was determined before hydrogel and antibiotic agents' addition and after 0.5, 1, 2, 4, 6, 24 and 48 h of incubation by a spectrophotometric assay. At each time point, both gentamicin and vancomycin showed only a partial inhibition of biofilm formation (ca. 30–40% for gentamicin; ca. 40–50% for vancomycin), with minor difference between the two studied microorganisms. On the other side, the hydrogel alone resulted in a significant reduction of biofilm of ca. 50%, in comparison to the untreated controls, while a combination of the hydrogel with either antibacterial coating resulted in a larger reduction of biofilm formation (approximately 75–80% in comparison with untreated controls).

Both these experimental studies show the ability of the DAC® hydrogel to significantly reduce bacterial adhesion and biofilm formation of common bacterial pathogens, thus potentially providing an effective protection of the implant; however, these data also point out how, in the clinical setting, in the absence of an adequate immune response from the host and/or of sufficient local levels of antibiotics, a passive antiadhesive coating [18] like HA can be overcome by the remaining bacteria in a time-dependent manner. For this reason, any passive antiadhesive coating of implants [53] should probably better be seen as a tool to reduce and delay bacterial adhesion and biofilm formation to a variable degree, also depending on the local environment, the contaminating bacterial species and initial bacterial load; this activity of the coating may represent a key additional advantage to the host's cells to win the competition with the microorganisms that may eventually be present. However, the known intrinsic limits of all passive coatings ground the idea of adding antibacterial agents to the protective hydrogel, in order to minimize the possibility for planktonic bacteria, which may eventually remain in the local environment, to colonize the implant at a second stage, when the coating has been hydrolyzed or covered by host's proteins.

#### **5.3. Antibiotic release studies**

Although designed as a "stand alone" product, the DAC® hydrogel was also tested concerning its ability to entrap and eventually release locally various antibacterial agents. As outlined above, the rationale for this combination lies in the pathogenesis of implant-related infections and on the specificities of passive protective coatings. In fact, biofilm formation is a multistep process that schematically includes the bacterial adhesion to a substrate, the subsequent immediate release of signals from adherent bacteria that triggers biofilm production and, finally, the biofilm construction and progressive consolidation. Acting mainly as a physical and antiadhesive barrier, DAC® hydrogel may reduce or prevent the first phase of the process, provided that the number of living bacteria is not too high and that they are not able to overcome or hydrolyze the hydrogel [54]; moreover, for an effective prevention of bacterial colonization of the coated implant, it is necessary that, while bacteria are in the more vulnerable planktonic state, they are completely removed or killed by the host's immune system and/or by the local chemicophysical environment. This is why, even in the presence of DAC® coating protection, systemic antibiotic prophylaxis is still to be considered necessary. In fact, if not eliminated, the remaining floating microorganisms may successfully colonize the implant once the protective coating has been hydrolyzed (a phenomenon that is expected to happen normally within 3 days from application for the DAC hydrogel) or after the implanted biomaterial has been covered by host's proteins (fibrin, fibronectin, etc.), which may also work as suitable for bacterial adhesion. In this scenario, the possibility to add also an antibacterial drug to the coating may further contribute to reduce the planktonic microorganisms, enhancing the overall protection offered by the DAC hydrogel (**Figure 4**).

Reductions of adhered bacteria on sterile titanium discs, coated with DAC® hydrogel, equal to 86.8, 80.4, 74.6 and 66.7% vs. untreated discs were observed after 15, 30, 60 and 120 min of incubation, respectively [37]. In another experiment, the ability to dislodge previously adhered bacteria was investigated. Once again, the results showed that DAC® hydrogel treatment of discs reduced the amount of adhered bacteria in respect to control discs after 15, 30,

Concerning more specifically the antibiofilm activity, DAC® hydrogel showed similar or superior *in vitro* activity, compared to various antibacterials and a synergistic activity when used in combination [48]. In one experimental setting, *S. epidermidis* and *S. aureus* were grown on chrome-cobalt devices in 6-wells polystyrene plates containing TSB for 24 h at 37°C. The plates were incubated at 37°C in ambient air, until a visible biofilm was obtained. Gentamycin and vancomycin were tested at a final concentration of 20 mg/mL. Similarly, when mixed with the hydrogel, 60 mg of gel powder was reconstituted with 1 mL of water for injections containing gentamicin or vancomycin at 20 mg/mL concentration. The amount of biofilm at each time was determined before hydrogel and antibiotic agents' addition and after 0.5, 1, 2, 4, 6, 24 and 48 h of incubation by a spectrophotometric assay. At each time point, both gentamicin and vancomycin showed only a partial inhibition of biofilm formation (ca. 30–40% for gentamicin; ca. 40–50% for vancomycin), with minor difference between the two studied microorganisms. On the other side, the hydrogel alone resulted in a significant reduction of biofilm of ca. 50%, in comparison to the untreated controls, while a combination of the hydrogel with either antibacterial coating resulted in a larger reduction of biofilm formation

Both these experimental studies show the ability of the DAC® hydrogel to significantly reduce bacterial adhesion and biofilm formation of common bacterial pathogens, thus potentially providing an effective protection of the implant; however, these data also point out how, in the clinical setting, in the absence of an adequate immune response from the host and/or of sufficient local levels of antibiotics, a passive antiadhesive coating [18] like HA can be overcome by the remaining bacteria in a time-dependent manner. For this reason, any passive antiadhesive coating of implants [53] should probably better be seen as a tool to reduce and delay bacterial adhesion and biofilm formation to a variable degree, also depending on the local environment, the contaminating bacterial species and initial bacterial load; this activity of the coating may represent a key additional advantage to the host's cells to win the competition with the microorganisms that may eventually be present. However, the known intrinsic limits of all passive coatings ground the idea of adding antibacterial agents to the protective hydrogel, in order to minimize the possibility for planktonic bacteria, which may eventually remain in the local environment, to colonize the implant at a second stage, when the coating has been hydrolyzed or covered by host's proteins.

Although designed as a "stand alone" product, the DAC® hydrogel was also tested concerning its ability to entrap and eventually release locally various antibacterial agents. As outlined above, the rationale for this combination lies in the pathogenesis of implant-related infections and on the specificities of passive protective coatings. In fact, biofilm formation is a multistep

60 and 120 min by 84.0, 72.8, 72.3 and 64.3%, respectively [37].

186 Hydrogels

(approximately 75–80% in comparison with untreated controls).

**5.3. Antibiotic release studies**

To test the hydrogel ability to entrap antibacterial drugs, vancomycin and tobramycin had been originally chosen as examples of antibiotic molecules [24]. Both these antibiotics have been added to the hydrogel just before its use, a solution that offers several advantages. First of all, this allows to add the antibiotic when it is needed, thus avoiding the problems of shelflife and any long-term compatibility with the hydrogel; secondly, in this way, it is possible to choose the optimal antibiotic in the specific case, taking into account the patient's specificity (e.g., known intolerances to specific antibacterial agents) or of the specific intervention; finally, the dosing of the antibacterial agent on a case-by-case basis can be decided by the clinician. The results of the *in vitro* study clearly showed how the investigated antibacterial hydrogel coating, applied on a titanium disc, at a concentration in polymer in the range 2–8% (w/v) and a concentration in drug equal to 1 or 2% (w/v) is able to release vancomycin or tobramycin, or of their association, for up to 72 h, with an amount of drug released that is hundred or thousand times higher than the minimum inhibitory concentration (MIC), in a time- and dose-dependent manner.

Similar results were founded by testing several other antibacterial compounds or their combinations, including vancomycin, teicoplanin, rifampicin, daptomycin, tigecycline, cefazolin, gentamicin, tobramycin, amikacin, meropenem, levofloxacin, etc. (cf. **Figure 5**), at concentrations ranging from 2 to 10% [48].

In summary, all examples reported above show that DAC® hydrogel is potentially able to entrap and release suitable quantities of antibacterial agents just after the implant of the coated prosthesis. The high initial burst effect of the released drug may ensure the most efficient action at the time that is critical for the destiny of the biomaterial. Moreover, during the entire drug release period, the antibiotic concentration released by the hydrogel remains greater than MIC, thus further ensuring effectiveness of the drug released in proximity to the prosthesis.

scanning electron microscopy (SEM) analysis [37]. This is an important requirement in order to reduce the exposed surface of a biomaterial, thus creating a uniform coating of the surface and leaving no pores or cracks that could eventually be colonized by planktonic bacteria.

**Figure 5.** Tigecycline-loaded DAC® hydrogel coating, applied at surgery on a knee revision prosthesis. The hydrogel, which comes in a powder form, in a prefilled syringe, is designed to be reconstituted at the time of surgery with water for injection. The surgeon may decide to add a single or a combination of antibiotics to the water for injection, to further

Hyaluronic-Based Antibacterial Hydrogel Coating for Implantable Biomaterials in Orthopedics…

http://dx.doi.org/10.5772/intechopen.73203

189

The resistance to scraping and declothing has also been tested in the animal model and in human femurs, simulating a press-fit insertion of a cementless implant [24, 48]. Both studies demonstrated the ability of the hydrogel coating to resist insertion, with 60% to more than 80% of the hydrogel remaining adherent to the entire implant surface, while the remainder

Safety and efficacy of the DAC® hydrogel have been investigated in several animal studies.

Concerning efficacy, in an acute model of highly contaminated implant-related infection in the rabbit, Giavaresi and coworkers [55] found that a vancomycin-loaded DAC® coating was associated with local bacterial load reduction ranging from 72 to 99%, compared to uncoated controls. In another large investigation in the rabbit model, Boot et al. [56] showed, at longer follow-up and without systemic antibiotic prophylaxis, the ability of vancomycin-loaded DAC®-coated implants to significantly resist infection, compared to uncoated controls. Both studies did also reveal the absence of local or systemic side effects. In line with this observation, a more

being retrieved along the inner surface of the medullary canal.

**6. DAC® hydrogel** *in vivo* **activity**

enhance implant protection.

**Figure 4.** Rationale for intra-operative mixing of DAC® hydrogel coating with antibacterial agents. Schematic representation of different scenarios. (a) Noncoated implants may get colonized by biofilm-forming bacteria (yellow circles) and infection will develop. (b) Antiadhesive coating may reduce/prevent bacterial adhesion, while the immune system (orange circles and red stars) and the systemically administered antibiotics (blue star) kill planktonic microorganisms. (c) However, if bacterial load is large enough, or if immune response and local antibiotic levels are inadequate, surviving bacteria may eventually colonize the implant, once the coating has been hydrolyzed or covered by host's proteins. (d) To prevent this, the antibacterial hydrogel may be loaded, at the time of surgery, with antibiotic agents (blue stars) that may be locally released, contributing to eliminate all remaining planktonic bacteria.

#### **5.4. DAC® hydrogel coating ability**

For any device candidate to act as a coating of orthopedic and trauma biomaterials, mechanical adherence to the implant surface plays a key role. In particular, DAC® hydrogel has been designed to be spread manually at the time of surgery and to not interfere with the usual surgical techniques of press-fit insertion of an implant. The ability of DAC® hydrogel to completely cover even sand-blasted titanium surface and resist scraping has been confirmed by

**Figure 5.** Tigecycline-loaded DAC® hydrogel coating, applied at surgery on a knee revision prosthesis. The hydrogel, which comes in a powder form, in a prefilled syringe, is designed to be reconstituted at the time of surgery with water for injection. The surgeon may decide to add a single or a combination of antibiotics to the water for injection, to further enhance implant protection.

scanning electron microscopy (SEM) analysis [37]. This is an important requirement in order to reduce the exposed surface of a biomaterial, thus creating a uniform coating of the surface and leaving no pores or cracks that could eventually be colonized by planktonic bacteria.

The resistance to scraping and declothing has also been tested in the animal model and in human femurs, simulating a press-fit insertion of a cementless implant [24, 48]. Both studies demonstrated the ability of the hydrogel coating to resist insertion, with 60% to more than 80% of the hydrogel remaining adherent to the entire implant surface, while the remainder being retrieved along the inner surface of the medullary canal.
