**1. Introduction**

Important new advances have been reported about new materials and biomaterials [1]. In the field of biomaterials, polymers and copolymers have found large application in modern medicine [2]. Biodegradability of polymeric biomaterials constituted a significant advantage, being that these materials are able to be broken down and removed when they have exerted their function [2]. Clinically, there are a wide range of applications of degradable polymers, such as surgical sutures and implants. Desired physical, chemical, biological, biomechanical, and degradation properties can be selected and tuned in order to fit all the requirements of the functional demand.

Fortunately, novel materials are developed constantly to meet new challenges providing a growing number of natural and synthetic degradable polymers, investigated for biomedical applications. Particularly, degradable polymers are of interest since these biomaterials are able to be molecularly broken down and eliminated or resorbed without physical removal or surgical revision [3].

In the design of biodegradable biomaterials, great importance should be given to their properties [3]. In fact, these materials should not produce a sustained inflammatory response and have a degradation

time coinciding with their function. Moreover, they should have appropriate mechanical properties for the scope, produce non-toxic degradation products that can be readily resorbed or excreted, and include appropriate permeability and processability for the designed application [3].

Orthopedic and traumatology (O&T) are up to 38% of the worldwide leading markets of implanted biomaterials [4], involving each year millions of new patients with an increasing trend. Infections related to implanted medical devices depend on the bacterial capability to establish highly structured multilayered biofilms on artificial surfaces and represent the most devastating complication in O&T, with millions of cases. In many cases, a slow release of antibiotic [5] can help the success of the intervention.

The aim of the present work is to assess the stability and the molecular degradation of a disposable coating of the implanted biomaterial Defensive Antibacterial Coating (DAC). Produced in the form of a powder, this device must be hydrated with water for its preparations, and is injectable alone or in solution, which is associated with an antibiotic to obtain a formulation in a hydrogel. The specific indication is for the prevention of peri-implant infection. This device, based on a novel resorbable hydrogel, would act as a resorbable barrier delivering local antibiofilm and antibacterial compounds. The active drugs will be mixed at the same time as the application of the hydrogel during surgery, allowing the correct choice for any given patient, reducing costs and improving storage life and versatility of use. This hydrogel is a derivative of hyaluronic acid (HA) which is present in large quantity in synovial fluid and vitreous humor, which contributes to these tissues' viscoelastic properties and which plays an important structural role in articular cartilage. HA is a natural linear polymer with disaccharide repeating units, namely, [(1→4)-beta-d-GlcpA-(1→3)-beta-d-GlcpNAc]. Natively, HA has been extracted from a variety of animal tissues in the past. Due to the limited sources and a risk of viral infection, the extraction technique has been replaced by microbial fermentation with a high purification efficiency, low production cost, and low risk rate of cross-species viral infection [6–9]. Owing to its relevantly unique physico-chemical properties and its important biological activities as a drug carrier [10–12], HA has been used in a wide variety of applications, such as food, biomedicine, biomaterials, and cosmetics. The gels obtainable by using cross-linked HA chains by chemical derivatization led to several chemical modifications. The cross-linking has been proposed to achieve chemical and mechanical HA robustness [13–15]. In particular, the conjugation with poly-D,L-lactic acid (PLA) [4,16–19] (Figure 1) gives an ideal product for the predesigned pharmacological use. → →

**Figure 1.** The molecular moieties (**a**) hyaluronic acid (HA) and (**b**) polylactic acid (PLA) forming the copolymer HA-PLA (b/a + b = 13% *w*/*w*).

In fact, this HA derivative is important for two main requisites to be reached. First, the stability of the hyaluronic acid chain against depolymerization, and secondly, the stability of the chemical junction between HA polymer and PLA moiety to confer to the polymer the desired physico-chemical characteristics. The sterilization procedure by ionizing through gamma ray irradiation induces a fragmentation of HA, as has been well reported [20]. The samples for the stability study have been synthesized by a new method property of Novagenit. The product has been named HA-PLA Novagenit

DAC® and obtained chemically in a sterile condition with an esterification of HA of about 13% (*w*/*w*) at the origin. The stability of the conjugate with sterile polylactic acid has been studied directly at the surface of titanium prosthesis as well as in the bulk region. For these samples, the physico-chemical characteristics, the stability of the copolymer, and its molecular weight dispersion have been studied by NMR spectroscopy [21] over time, which included a temperature cycle. The earlier proposed application of the diffusion-ordered spectroscopy (DOSY) NMR revealed it was particularly successful in this kind of study in order to distinguish among different molar diffusivities due to the different hydrodynamic radii that are often correlated with the different molecular weights [22–25]. In this field, very important advances have been made by DOSY NMR in general [26,27] for the separation of drugs in mixtures [28,29]. It is important that in similar studies addressed to the measure of precise molar diffusivity, should be performed usually with a gradient strength quite higher than that used in this study. Thus, the reported results are to be considered as apparent values that, notwithstanding, allowed us to monitor the macromolecular degradation and the extent of the hydrolysis of the conjugation. The characteristic of linear polymers (far from the usual spherical shape of macromolecules) led us to consider these results valid [30]. A discussion of the obtained results is detailed in a paragraph in the discussion section.

This study appears particularly important because of the diverse biological activities of the hyaluronan fragments [31]. In fact, depending on the polymer length, i.e., small, medium, and large fragments, small and medium ones have pro-angiogenic and anti-apoptotic properties which stimulate the synthesis of heat-shock proteins (HSP) as potent immunostimulants. Immunosuppressive and anti-angiogenic functions are mainly exerted by large polymers [12].

As reference standards with high molecular homogeneity, certified HAs at various molecular weights (MWs) have been used. This approach was already applied in other cases to reveal the presence of hyaluronic acid in mixtures [30]. In our case, this spectroscopic approach revealed to be more precise and sensitive than the results obtained by gel permeation chromatography reported below [32,33].
