ib A Chitosan-based Hydrogel with PLCL, ZnO NPs, and Oligoelements: A Promising Antibiotic Scaffold for Tissue Engineering

Tissue engineering involves anchorage-dependent cells cultured on scaffolds, with growth factors added to facilitate cell proliferation. Its use in transplants implies the risk of bacterial infection. The current contribution describes the preparation and antibacterial evaluation of a chitosan-based hydrogel physically cross-linked with poly(l-lactic-co-ɛ -caprolactone) (PLCL) and enriched with zinc oxide nanoparticles (ZnO NPs) and trace elements (potassium and magnesium). The material was developed as a scaffold with built-in antibacterial properties. Chitosan and PLCL are biocompatible support materials applied in medicine for the repair and regeneration of damaged tissues, ob-jectives promoted by ZnO NPs and the aforementioned trace elements. The ZnO NPs were elaborated by chemical coprecipitation. The materials were characterized by XRD, FT-IR, and SEM. Antibacterial testing was performed with strains of Escherichia coli and Staphylococcus aureus by the Kirby-Bauer method, in accordance with the NCCLS and CLSI guidelines. It was possible to obtain a homogeneous hydrogel with adequate morphology and distribution of elements. The hydrogel with 300 mM of Mg, K, and ZnO NP’s showed antibacterial inhibition halos of 13 mm for S. aureus and 19 mm for E. coli . This innovative biomaterial with trace elements holds promise for tissue engineering by considering the challenge of bacterial infection.


INTRODUCTION
Tissue engineering aims to establish, restore, or increase the function of tissues by means of the in vitro culturing of anchorage-dependent cells (either differentiated or undifferentiated) on scaffolds made of biomaterials. Growth factors are added to facilitate cell proliferation.
The in vitro tissues are then transplanted to a target organ [1] [2] with tissue injury. Such an injury is susceptible to infection by pathogenic microorganisms, which can lead to the loss or alteration of transplanted tissue, thus complicating tissue recovery and sometimes causing implant failure [1] .
The biomaterials utilized in tissue engineering are biocompatible polymers, such as chitosan, that elicit robust cell proliferation [3] . The latter biopolymer is biocompatible, biodegradable, non-toxic, non-mutagenic, bioactive, cationic, antibacterial, and antifungal. It has been of great interest in developing drug-delivery scaffolds for tissue engineering and has been classified as "generally recognized as safe" (GRAS) by the US Food and Drug Administration (FDA) [4] [5] .
Another material approved by the FDA to foster cell proliferation and adhesion is the poly(l-lactide-co-ɛcaprolactone) (PLCL) polymer, due to its biocompatibility and mechanical properties [6] .
Chitosan can be combined with certain trace elements essential for biological, physiological, and enzymatic processes. Consequently, zinc (Zn), potassium (K), and magnesium (Mg) [7] are added to improve certain functions, including protection against bacteria or fungi, bone regeneration, the synthesis of proteins, the absorption of Fe3+, the protection of tooth enamel, and the production of skin collagen [8] [9] [10] . ZnO nanoparticles (NPs), on the other hand, promote cell proliferation, growth, and differentiation. Additionally, their nanometric size affords high reactivity and thus favors an antibacterial effect [11] .
During a surgical intervention for organ or tissue implantation, the main agents of infection are bacteria, which are able to generate septicemia, impetigo, cellulitis, skin abscesses, and even patient death [12] .
The combination of chitosan, PLCL, ZnO NPs, and trace elements has an exciting future in tissue engineering. Apart from biocompatibility, this complex has the capacity for cell proliferation as well as inhibition of infection by nosocomial pathogens (e.g., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens) in surgical incisions and other medical conditions (e.g., diabetic foot and venous ulceration) [13] .
The aim of the present study was to develop an HQT hydrogel with chitosan physically cross-linked to PLCL, with the addition of ZnO NPs and trace amounts of K and Mg. The ZnO NPs were obtained by chemical coprecipitation. The HQT hydrogel and its components were characterized with X-ray diffraction (XRD), Laboratory Standards (NCCLS) [14] and the Clinical & Laboratory Standards Institute (CLSI) [15] .

a) Synthesis of ZnO NPs
Nanoparticles were synthesized by the coprecipitation method.    [16] . The size of the crystallites was 30 nm, calculated with the Scherrer equation. No secondary phases were formed during synthesis.    EDS spectra (b), and elemental mapping (c, d, e, and f). The FT-IR spectra are shown for trace elements, PLCL, and the hydrogel (Figure 3). All samples were dissolved in 1 M acetic acid. The vibrational bands of ~3398-3300 cm -1 and 1637 cm -1 in spectra 1, 2, and 3 can be attributed to the OH group in the solvent [17] . The spectrum of chitosan (Figure 3.4) exhibits a band at ~3323 cm -1 for the OH/NH groups, at ~1644 cm -1 for C-N, at ~1392 cm -1 for C=O, and at ~1288 cm -1 for C-O-C (corresponding to amine 1), C-H vibrations, and C-C and C-O stretching (corresponding to amine 3), respectively [18] [19] .
The main contributions of PLCL are seen in Figure 3.5.
The bands at ~1380 cm -1 were ascribed to C=O, at ~1189 cm -1 and ~1182 cm -1 to C-O-O and asymmetric CH 2 , and at ~1091 cm -1 to the stretching vibrations of C-O [20] . The FT-IR absorption bands of the HQT hydrogel (with all its components) are shown in Figure 3.6, observing a clear difference in the interaction of the functional groups. The bands at ~3300 cm -1 were designated as the NH 2 and OH groups, at ~1750 cm -1 as the stretching of the carbonyl groups in PLCL, at ~1600 cm -1 as the CH 2 and CH 3 groups, and at ~1250 cm -1 as a stretching of the C-O-C present in chitosan. Finally, the band at ~1420 cm -1 may be due to the chitosan CH 2 groups or PLCL-COO-groups [18] [19] [20] .

b) Antibacterial activity
The antibacterial effect of the HQT hydrogel was fomenting its disintegration or rupture [21] .
Additionally, the size of ZnO NPs is effective against Gram-positive and Gram-negative bacteria because of the large surface area available to interact with the bacterial wall [22] .
For the biomaterials with trace elements, the inhibition of E. coli was more significant than that of S.
aureus. The content of K and Mg affect the antibacterial activity of ZnO NPs, as can be appreciated by the lack of inhibition of both microorganisms. Concerning E. coli, the inhibition diameters resulting from K combined with Q (Q-K) materials were very similar to those found with chitosan alone (14 mm). The antibacterial effect of Q-K materials on S. aureus was reduced by about 50% (to 12 mm) (Figure 4). K is required by microorganisms for the metabolism of carbohydrates, the activation of some enzymes, and bacterial osmoregulation [23] . Therefore, it is herein inferred that K was mainly used as a nutrient by the bacteria rather than a growth inhibitor. For E. coli, the inhibition diameter was greater with Q-Mg (17 mm) than chitosan alone (14 mm). Thus, Mg increased the antibacterial potential of chitosan. Mg and K did not show any antibacterial activity separately. They participate in vital functions as nutrients [23] , which was  to evaluate the inhibitory effect of the present biomaterials on both species [13] . According to the literature, chitosan, PLCL and ZnO NPs are non-toxic materials and have been used in various biomedical applications. The current results indicate that combining biomaterials on a chitosan hydrogel scaffold holds promise for combatting tissue infection caused by bacteria.
Future research is necessary to examine the cytotoxicity of the hydrogel.