**4. Synthesis of DAC® HA-g-PLA hydrogel coating**

rhinosinusitis, whether or not associated with polyposis, is well documented, as well as results

HA has also been reported to exert bacteriostatic, dose-dependent effect on different planktonic microorganisms [32, 33]. Radaeva et al. showed the inhibiting activity of HA with respect to some *Pseudomonas* species [34], while Ardizzoni and coworkers [23] investigated the effects of HA on 15 ATCC bacterial strains, representative of clinically relevant bacterial and fungal species. According to their results, different microbial species and strains are differently affected by HA. In particular, staphylococci, enterococci, *Streptococcus mutans*, two *Escherichia coli* strains, *Pseudomonas aeruginosa*, *Candida glabrata* and *C. parapsilosis* showed a dose-dependent growth inhibition, while no HA effects were detected in *E. coli* ATCC 13768

Carlson and coworkers [33] compared the potential bacteriostatic effect of collagen type I, hyaluronic acid, hydroxyapatite, polylactic acid and polyglycolic acid on some of the most common orthopedic bacterial pathogens (*S. aureus*, *S. epidermidis*, *β-hemolytic Streptococcus* and *Pseudomonas aeruginosa*): HA had the most significant bacteriostatic properties on the studied organisms. Similarly, Pirnazar et al. [32] did demonstrate the bacteriostatic effect of HA in different concentrations and molecular weight on oral and nonoral microorganisms (*Staphylococcus aureus*, *Propionibacterium acnes*, *Actinobacillus actinomycetemcomitans*, *Prevotella oris* and *Porphyromonas gingivalis*). The authors concluded that the clinical application of hyaluronan in the form of membranes, gels or sponges may reduce bacterial contamination of the surgical wound, thereby lessening the risk of postsurgical infection and promoting more predictable regeneration.

Concerning orthopedic applications, Harris and Richards [35] showed how coating titanium with sodium hyaluronate significantly decreased the density of *S. aureus* adhering to the surfaces and proposed its potential use to protect osteosynthesis, orthopedic or dental implants. In a recent review, focused on the use of polysaccharide-based coatings to prevent biofilm formation, hyaluronic acid was discussed as one of the most promising [36]; displaying hydrophilic characteristics, this coating was in fact reported to reduce adhesion of *S. aureus*, *S. epidermidis* and *E. coli* by several orders of magnitude compared to unmodified surfaces.

Several clinical local applications of HA to reduce the impact of biofilm-related infections

Torretta et al. [38] recently described topical administration of hyaluronic acid in children

Other studies have documented the positive effect of topical HA in chronic urinary tract infections (UTI). At variance with current antibiotic treatments, aimed at eradicating pathogens, HA local administration targets bacterial adherence to the bladder mucosa [39–42]. Damiano et al., in a prospective, randomized, double-blind, placebo-controlled study, showed a significant reduction of 77% (P < 0.0002) in the UTI rate per year in HA-treated patients, compared to controls. Moreover, mean time to UTI recurrence was significantly prolonged (185.2 ± 78.7

**3. Clinical applications of HA to prevent bacterial adhesion**

with recurrent or chronic middle ear inflammations and chronic adenoiditis.

have been reported with favorable results and no adverse events [37].

from its effects on mucociliary clearance, free radical production and mucosal repair."

and *C. albicans*, and *S. sanguinis* was favored by the highest HA dose.

182 Hydrogels

Composed of covalently linked hyaluronan and poly-d,l-lactide, the "Defensive Antibacterial Coating" (DAC®, Novagenit Srl, Mezzolombardo, Italy) was specifically developed in order to protect implanted biomaterials used in orthopedics, traumatology, dentistry and maxillofacial surgery from bacterial colonization [24, 48] (**Figure 2**).

**Figure 2.** DAC® HA-g-PLA, fast-resorbable, hydrogel coating. Composed of covalently linked hyaluronan and poly-d, l-lactide, the "Defensive Antibacterial Coating" (DAC®, Novagenit Srl, Mezzolombardo, Italy) is the first antibacterialcoating cleared for clinical use in orthopedics, trauma, dentistry and maxillofacial surgery in Europe.

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

*δ* 2.80 (m, 4H, ─OC─CH2

1H, ─O─CO─**CH**(CH3

PLA), 1382 (*δ* as CH3

*δ* 1.25 and *δ* 1.45 (2d, ─O─CO─CH(CH3

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

toxicity or interference with bone or periimplant tissues.

class III medical devices is largely accepted and tested safe.

**5.2. Antiadhesive and antibiofilm activity**

formation were extensively studied *in vitro*.

5.1 ppm (m, ─O─CO─CH(CH3

**5.1. Cell compatibility assay**

─CH2

holic and ether of HA) cm−1. 1H NMR of HA-g-PLA (DMSO-*d*<sup>6</sup>

to N-acetylglucosamine residue of HA and resulted to be 7 ± 1 mol%.

─CO─); *δ* 4.3 and *δ* 5.2 (m, 1H, ─O─CO─**CH**(CH3

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

)─O). The synthesis of HA-g-PLA copolymer was carried out as follows:

of PLA), 1189 (*ν* as C─O─C ester group of PLA), 1089, 1048 (*ν* C─O alco-

/D2

)─O─ of PLA); *δ* 1.85 (s, 3H, ─NH─CO─CH3

)─ of PLA). The % degree of grafting (DG) has been calculated

600 mg of HA-TBA was dissolved in 48 ml of anhydrous dimethyl sulfoxide (DMSO) and then 576 μl of DEA, as a catalyst, was added. A suitable amount of PLA-NHS (dissolved in 6 ml of anhydrous DMSO) was added according to *X* = 1, *X* being equal to moles of PLA-NHS/ moles of HA repeating units. The PLA-NHS solution was added drop by drop to the HA-TBA solution in about 1 h. The reaction was carried out under argon at 40°C for 24 h. After this time, the TBA was exchanged with Na<sup>+</sup> using a Dowex 50 W × 8-200 resin, and then the eluate was dialysed against distilled water, by using spectra/por tubing with a cutoff of 14,000 Da and then freeze-dried. The sample has been characterized by FT-IR and 1H NMR analyses. FT-IR spectrum (KBr) of HA-g-PLA showed a broad band centered at 3450 cm−1 (*ν* as OH + *ν* as NH of HA), bands at 1757 (*ν* as COO of PLA), 1623 (amide I of HA), 1456 (*δ* as CH3

as: %DG = (moles PLA chains/moles of HA repeating units) × 100. The degree of grafting was determined by comparing the integral of the peaks at *δ* 1.25–1.45 attributed to protons of methyl

*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

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

Both the ability of the DAC® HA-g-PLA hydrogel to reduce bacterial adhesion and biofilm

groups of PLA with the integral of peaks at *δ* 1.85 attributed to protons of NHCO**CH<sup>3</sup>**

)─OH; m,

185

of

of HA) *δ*

belonging

O 90:10) spectrum showed:

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

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 (CDCl3 ) showed: *δ* 1.5 and *δ* 1.6 (d, 3H, ─O─CO─CH(CH3 )─OH; d, 3H, ─O─CO─CH(CH3 )─O─),

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

*δ* 2.80 (m, 4H, ─OC─CH2 ─CH2 ─CO─); *δ* 4.3 and *δ* 5.2 (m, 1H, ─O─CO─**CH**(CH3 )─OH; m, 1H, ─O─CO─**CH**(CH3 )─O). The synthesis of HA-g-PLA copolymer was carried out as follows: 600 mg of HA-TBA was dissolved in 48 ml of anhydrous dimethyl sulfoxide (DMSO) and then 576 μl of DEA, as a catalyst, was added. A suitable amount of PLA-NHS (dissolved in 6 ml of anhydrous DMSO) was added according to *X* = 1, *X* being equal to moles of PLA-NHS/ moles of HA repeating units. The PLA-NHS solution was added drop by drop to the HA-TBA solution in about 1 h. The reaction was carried out under argon at 40°C for 24 h. After this time, the TBA was exchanged with Na<sup>+</sup> using a Dowex 50 W × 8-200 resin, and then the eluate was dialysed against distilled water, by using spectra/por tubing with a cutoff of 14,000 Da and then freeze-dried. The sample has been characterized by FT-IR and 1H NMR analyses. FT-IR spectrum (KBr) of HA-g-PLA showed a broad band centered at 3450 cm−1 (*ν* as OH + *ν* as NH of HA), bands at 1757 (*ν* as COO of PLA), 1623 (amide I of HA), 1456 (*δ* as CH3 of PLA), 1382 (*δ* as CH3 of PLA), 1189 (*ν* as C─O─C ester group of PLA), 1089, 1048 (*ν* C─O alcoholic and ether of HA) cm−1. 1H NMR of HA-g-PLA (DMSO-*d*<sup>6</sup> /D2 O 90:10) spectrum showed: *δ* 1.25 and *δ* 1.45 (2d, ─O─CO─CH(CH3 )─O─ of PLA); *δ* 1.85 (s, 3H, ─NH─CO─CH3 of HA) *δ* 5.1 ppm (m, ─O─CO─CH(CH3 )─ of PLA). The % degree of grafting (DG) has been calculated as: %DG = (moles PLA chains/moles of HA repeating units) × 100. The degree of grafting was determined by comparing the integral of the peaks at *δ* 1.25–1.45 attributed to protons of methyl groups of PLA with the integral of peaks at *δ* 1.85 attributed to protons of NHCO**CH<sup>3</sup>** belonging to N-acetylglucosamine residue of HA and resulted to be 7 ± 1 mol%.
