Implants coating strategies for antibacterial treatment in fracture and defect models: A systematic review of animal studies.

Objective: Fracture-related infection (FRI) remains a major concern in orthopaedic trauma. Functionalizing implants with antibacterial coatings are a promising strategy in mitigating FRI. Numerous implant coatings have been reported but the preventive and therapeutic effects vary. This systematic review aimed to provide a comprehensive overview of current implant coating strategies to prevent and treat FRI in animal fracture and bone defect models. Methods: A literature search was performed in three databases: PubMed, Web of Science and Embase, with predetermined keywords and criteria up to 28 February 2023. Preclinical studies on implant coatings in animal fracture or defect models that assessed antibacterial and bone healing effects were included. Results: A total of 14 studies were included in this systematic review, seven of which used fracture models and seven used defect models. Passive coatings with bacteria adhesion resistance were investigated in two studies. Active coatings with bactericidal effects were investigated in 12 studies, four of which used metal ions including Ag+ and Cu2+; five studies used antibiotics including chlorhexidine, tigecycline, vancomycin, and gentamicin sulfate; and the other three studies used natural antibacterial materials including chitosan, antimicrobial peptides, and lysostaphin. Overall, these implant coatings exhibited promising efficacy in antibacterial effects and bone formation. Conclusion: Antibacterial coating strategies reduced bacterial infections in animal models and favored bone healing in vivo. Future studies of implant coatings should focus on optimal biocompatibility, antibacterial effects against multi-drug resistant bacteria and polymicrobial infections, and osseointegration and osteogenesis promotion especially in osteoporotic bone by constructing multi-functional coatings for FRI therapy. The translational potential of this paper: The clinical treatment of FRI is complex and challenging. This review summarizes novel orthopaedic implant coating strategies applied to FRI in preclinical studies, and offers a perspective on the future development of orthopaedic implant coatings, which can potentially contribute to alternative strategies in clinical practice.

Active Osteoblasts or Quiescent Bone Lining Cells? Preosteoblasts Fate Orchestrated by Curvature and Stiffness of an In Vitro 2.5D Biomimetic Culture System.

Biomimetic cell culture systems are required to provide more physiologically relevant microenvironments for bone cells. Here, a simple 2.5D culture platform is proposed, combining adjustable stiffness and surface features that mimic bone topography by using sandpaper grits as master molds with two stiffness formulations of polydimethylsiloxane (PDMS). The subsequent replicas perfectly conform the grits and reproduce the corresponding negative relief with cavities separated by convex edges. Biomimicry is also provided by an extracellular matrix (ECM)-like thin film coating, using the layer-by-layer (LbL) method. The topographical features, alternating concave, and convex structures drive preosteoblasts organization and morphology. Strikingly, curvature orchestrates the commitment of preosteoblasts, with i) maturation to active osteoblasts able to produce a dense collagenous matrix that ultimately mineralizes in the cavities, and ii) edges hosting quiescent cells that synthetize a very thin immature collagen layer with no mineralization. In summary, the present in vitro culture system model offers a cell-instructive 2.5D microenvironment that controls preosteoblasts fate, leading to two coexisting subpopulations: mature osteoblasts and bone lining cells (BLC). This promising culture system opens new avenues to advanced tissue-engineered modeling and can be applied to precellularized bone biomaterials.

Methacrylated Cellulose Nanocrystals as Fillers for the Development of Photo-Cross-Linkable Cytocompatible Biosourced Formulations Targeting 3D Printing.

Cellulose nanocrystals (CNCs) from cotton were functionalized in aqueous medium using methacrylic anhydride (MA) to produce methacrylated cellulose nanocrystals (mCNCs) with a degree of methacrylation (DM) up to 12.6 ± 0.50%. Dispersible as-prepared CNCs and mCNCs were then considered as reinforcing fillers for aqueous 3D-printable formulations based on methacrylated carboxymethylcellulose (mCMC). The rheological properties of such photo-cross-linkable aqueous formulations containing nonmodified CNCs or mCNCs at 0.2 or 0.5 wt% in 2 wt% mCMC were fully investigated. The influence of the presence of nanoparticles on the UV-curing kinetics and dimensions of the photo-cross-linked hydrogels was probed and 13C CP-MAS NMR spectroscopy was used to determine the maximum conversion ratio of methacrylates as well as the optimized time required for UV postcuring. The viscoelasticity of cross-linked hydrogels and swollen hydrogels was also studied. The addition of 0.5 wt% mCNC with a DM of 0.83 ± 0.040% to the formulation yielded faster cross-linking kinetics, better resolution, more robust cross-linked hydrogels, and more stable swollen hydrogels than pure mCMC materials. Additionally, the produced cryogels showed no cytotoxicity toward L929 fibroblasts. This biobased formulation could thus be considered for the 3D printing of hydrogels dedicated to biomedical purposes using vat polymerization techniques, such as stereolithography or digital light processing.

Dual-functional antibacterial and osteogenic nisin-based layer-by-layer coatings.

Implanted biomaterials can be regarded in a cornerstone in the domain of bone surgery. Their surfaces are expected to fulfil two particular requirements: preventing the settlement and the development of bacteria, and stimulating bone cells in view to foster osseointegration. Therefore, a modern approach consists in the design of dual functional coatings with both antibacterial and osteogenic features. To this end, we developed ultrathin Layer-by-Layer (LbL) coatings composed of biocompatible polyelectrolytes, namely chondroitin sulfate A (CSA) and poly-l-lysine (PLL). The coatings were crosslinked with genipin (GnP), a natural and biocompatible crosslinking agent, to increase their resistance against environmental changes, and to confer them adequate mechanical properties with regards to bone cell behaviors. Antibacterial activity was obtained with nisin Z, an antimicrobial peptide (AMP), which is active against gram-positive bacteria. The coatings had a significant bactericidal impact upon Staphylococcus aureus, with fully maintained bone cell adhesion, proliferation and osteogenic differentiation.

Nisin-based antibacterial and antiadhesive layer-by-layer coatings.

Some removable medical devices such as catheters and cardiovascular biomaterials require antiadhesive properties towards both prokaryotic and eukaryotic cells in order to prevent the tissues from infections upon implantation and, from alteration upon removal. In order to inhibit cell adhesion, we developed ultrathin hydrated Layer-by-Layer (LbL) coatings composed of biocompatible polyelectrolytes, namely chondroitin sulfate A (CSA) and poly-l-lysine (PLL). The coatings were crosslinked with genipin (GnP), a natural and biocompatible crosslinking agent, to increase their resistance against environmental changes. In order to confer antibacterial activity to the coatings, we proceeded to the electrostatically-driven immobilization of nisin Z, an antimicrobial peptide (AMP) active against gram-positive bacteria. The nisin-enriched coatings had a significantly increased anti-proliferative impact on fibroblasts, as well as a strong contact-killing activity against Staphylococcus aureus in the short and long term.

Biomimetic matrix for the study of neuroblastoma cells: A promising combination of stiffness and retinoic acid.

Neuroblastoma is the third most common pediatric cancer composed of malignant immature cells that are usually treated pharmacologically by all trans-retinoic acid (ATRA) but sometimes, they can spontaneously differentiate into benign forms. In that context, biomimetic cell culture models are warranted tools as they can recapitulate many of the biochemical and biophysical cues of normal or pathological microenvironments. Inspired by that challenge, we developed a neuroblastoma culture system based on biomimetic LbL films of physiological biochemical composition and mechanical properties. For that, we used chondroitin sulfate A (CSA) and poly-L-lysine (PLL) that were assembled and mechanically tuned by crosslinking with genipin (GnP), a natural biocompatible crosslinker, in a relevant range of stiffness (30-160 kPa). We then assessed the adhesion, survival, motility, and differentiation of LAN-1 neuroblastoma cells. Remarkably, increasing the stiffness of the LbL films induced neuritogenesis that was strengthened by the combination with ATRA. These results highlight the crucial role of the mechanical cues of the neuroblastoma microenvironment since it can dramatically modulate the effect of pharmacologic drugs. In conclusion, our biomimetic platform offers a promising tool to help fundamental understanding and pharmacological screening of neuroblastoma differentiation and may assist the design of translational biomaterials to support neuronal regeneration. STATEMENT OF SIGNIFICANCE: Neuroblastoma is one of the most common pediatric tumor commonly treated by the administration of all-trans-retinoic acid (ATRA). Unfortunately, advanced neuroblastoma often develop ATRA resistance. Accordingly, in the field of pharmacological investigations on neuroblastoma, there is a tremendous need of physiologically relevant cell culture systems that can mimic normal or pathological extracellular matrices. In that context, we developed a promising matrix-like cell culture model that provides new insights on the crucial role of mechanical properties of the microenvironment upon the success of ATRA treatment on the neuroblastoma maturation. We were able to control adhesion, survival, motility, and differentiation of neuroblastoma cells. More broadly, we believe that our system will help the design of in vitro pharmacological screening strategy.

Microgels Based on Carboxymethylpullulan Grafted with Ferulic Acid Obtained by Enzymatic Crosslinking in Emulsion for Drug Delivery Systems.

Carboxymethylpullulan (CMP) grafted with ferulic acid (FA) is crosslinked with laccase by the reverse water-in-oil emulsion technique (with sunflower oil) to obtain microgels with size from 40 to 200 µm. It is demonstrated that laccase activity and dispersion time have an impact on microgels' size. Fluorescence spectroscopy of different probes (e.g., pyrene, Nile red, and curcumin) shows the nonpolar characteristics of hydrophobic microdomains formed by the FA moieties and its dimers forming the crosslinking nodes. Encapsulation and release of curcumin or lidocaine used as drug models are studied in different buffers. Curcumin is well encapsulated but retained in microgels, while lidocaine is released at 65-70% in 2 h and 30 min in buffer simulating the gastrointestinal tract and at 75-85% in 1 h in acetate buffer pH 5.6 or phosphate-buffered saline (PBS) pH 6.9.

Biomimetic hydrogel by enzymatic crosslinking of pullulan grafted with ferulic acid.

A novel eco-friendly two-step synthesis process of neutral pullulan (PUL)-ferulic acid (FA) conjugates was reported in this work. Ferulic acid was first transformed to activated ferulate-imidazolide using N,N'-carbonyldiimidazole (CDI), a green activated reagent. Issued product was then reacted with pullulan. PUL-FA derivatives were characterized by FTIR and 1H NMR leading to substitution degrees (DS) between 0.02 and 0.1 (mol FA per mol PUL repeat unit). The study in dilute regime indicated an associative behavior with the presence of aggregate structures in solution due to the hydrophobic interactions between the grafted FA onto polysaccharide backbones. Laccase from Trametes versicolor was then used to crosslink polysaccharide chains to obtain biomimetic PUL-FA hydrogels. Gelling's kinetics were analyzed with rheology in dynamic mode showing the impact of laccase amount, DS and concentration. Mechanical and swelling properties appear related only to DS and concentration of PUL-FA products.

Hyaluronan-based hydrogels as versatile tumor-like models: Tunable ECM and stiffness with genipin-crosslinking.

Three-dimensional (3D) biomimetic cell culture platforms offer more realistic microenvironments that cells naturally experience in vivo. We developed a tunable hyaluronan-based hydrogels that could easily be modified to mimic healthy or malignant extracellular matrices (ECMs). For that, we pre-functionalized our hydrogels with an adhesive polypeptide (poly-l-lysine, PLL) or ECM proteins (type III and type IV collagens), naturally present in tumorous tissues, and next, we tuned their stiffness by crosslinking with gradual concentrations of genipin (GnP). Then, we thoroughly characterized our substrates before testing them with glioblastoma and breast cancer cells, and thereafter with endothelial cells. Overall, our hydrogels exhibited (a) increasing stiffness with GnP concentration for every pre-functionalization and (b) efficient enzyme resistance with PLL treatment, and also with type IV collagen but to a lesser extent. While PLL-treated hydrogels were not favorable to the culture of any glioblastoma cell lines, they enhanced the proliferation of breast cancer cells in a stiffness-dependent manner. Contrary to type III collagen, type IV collagen pre-treated hydrogels supported the proliferation of glioblastoma cells. The as-desired HA-based 3D tumor-like models we developed may provide a useful platform for the study of various cancer cells by simply tuning their biochemical composition and their mechanical properties.

Antioxidant properties and bioactivity of Carboxymethylpullulan grafted with ferulic acid and of their hydrogels obtained by enzymatic reaction.

Antioxidant and cytocompatible chemically modified polysaccharides and their hydrogels were obtained by a biomimetic approach. For this purpose, carboxymethylpullulan grafted with ferulic acid (CMP-FA) was firstly synthesized with different substitution degrees (DSFA). Their hydrogels were secondly obtained by enzymatic cross-linking with laccase. Hydrogel swelling has been found dependent on both DSFA and media ionic strength. The CMP-FA antioxidant properties were evaluated by the DPPH method and ABTS assays. The DPPH radical scavenging effect was high for CMP-FA solutions (80% after 30 min) and lower for the corresponding hydrogels (70% after 7 h). The antibacterial properties of ferulic acid and CMP-FA derivatives were tested against Staphylococcus aureus but the minimal inhibitory concentration of CMP-FA was not reached in the range of concentrations studied. Finally the CMP-FA derivatives showed no cytotoxicity towards mouse fibroblast cells.

Carboxymethylpullulan Grafted with Aminoguaiacol: Synthesis, Characterization, and Assessment of Antibacterial and Antioxidant Properties.

Aminoguaiacol, the aminated derivative of guaiacol, a natural phenolic compound, was chemically grafted onto a polysaccharide (carboxymethylpullulan, CMP) in the presence of the activator agent 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDCI). The grafted polysaccharides were characterized by FTIR and 1H NMR spectroscopy to confirm and quantify the grafting. All polysaccharide derivatives (grafting rates of aminoguaiacol between 16% and 58%) were soluble in water. Their physicochemical properties were studied in a dilute regime and a semidilute regime by light scattering, fluorescence, and rheology, showing associative properties with peculiar polysoap behavior. The antibacterial activities of the synthesized products against Staphyloccocus aureus were assessed using a counting method. The antioxidant activities of the derivatives were also highlighted using the α,α-diphenyl-ß-picrylhydrazyl (DPPH) method. Finally, the cytotoxicity of the derivatives was studied with fibroblast cells and they showed a very good cytocompatibility. Such polymers could be used to replace chemical preservatives in food and cosmetic aqueous formulations.

Synergistic influence of topomimetic and chondroitin sulfate-based treatments on osteogenic potential of Ti-6Al-4V.

We combined topographical and chemical surface modifications of Ti-6Al-4V (TA6V) to improve its osteogenic potential. By acid-etching, we first generated topomimetic surface features resembling, in size and roughness, bone cavities left by osteoclasts. Next, we coated these surfaces with biomimetic Layer-by-Layer films (LbL), composed of chondroitin sulfate A and poly-l-lysine that were mechanically tuned after a post-treatment with genipin. The structural impact of each surface processing step was thoroughly inspected. The desired nano/microrough topographies of TA6V were maintained upon LbL deposition. Whereas no significant promotion of adhesion and proliferation of MC3T3-E1 preosteoblasts were detected after independent or combined modifications of the topography and the chemical composition of the substrates, osteogenic maturation was promoted when both surface treatments were combined, as was evidenced by significant long-term matrix mineralization. The results open promising route toward improved osseointegration of titanium-based implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1988-2000, 2016.

Genipin-cross-linked layer-by-layer assemblies: biocompatible microenvironments to direct bone cell fate.

The design of biomimetic coatings capable of improving the osseointegration of bone biomaterials is a current challenge in the field of bone repair. Toward this end, layer-by-layer (LbL) films composed of natural components are suitable candidates. Chondroitin sulfate A (CSA), a natural glycosaminoglycan (GAG), was used as the polyanionic component because it promotes osteoblast maturation in vivo. In their native state, GAG-containing LbL films are generally cytophobic because of their low stiffness. To stiffen our CSA-based LbL films, genipin (GnP) was used as a natural cross-linking agent, which is much less cytotoxic than conventional chemical cross-linkers. GnP-cross-linked films display an original combination of microscale topography and tunable mechanical properties. Structural characterization was partly based on a novel donor/acceptor Förster resonance energy transfer (FRET) couple, namely, FITC/GnP, which is a promising approach for further inspection of any GnP-cross-linked system. GnP-cross-linked films significantly promote adhesion, proliferation, and early and late differentiation of preosteoblasts.

Protein-triggered instant disassembly of biomimetic Layer-by-Layer films.

Layer-by-Layer (LbL) coatings are promising tools for the biofunctionalization of biomaterials, as they allow stress-free immobilization of proteins. Here, we explore the possibility to immobilize phosvitin, a highly phosphorylated protein viewed as a model of bone phosphoproteins and, as such, a potential promotive agent of surface-directed biomineralization, into biomimetic LbL architectures. Two immobilization protocols are attempted, first, using phosvitin as the polyanionic component of phosvitin/poly-(L-lysine) films and, second, adsorbing it onto preformed chondroitin sulfate/poly-(L-lysine) films. Surprisingly, it is neither possible to embed phosvitin as the constitutive polyanion of the LbL architectures nor to adsorb it atop preformed films. Instead, phosvitin triggers instant massive film disassembly. This unexpected, incidentally detected behavior constitutes the first example of destructive interactions between LbL films and a third polyelectrolyte, a fortiori a protein, which might open a route toward new stimuli-responsive films for biosensing or drug delivery applications. Interestingly, additional preliminary results still indicate a promotive effect of phosvitin-containing remnant films on calcium phosphate deposition.

Bone remodeling regulation under unloading conditions: numerical investigations.

The present paper addresses the following question: can a simple regulatory bone remodeling model predict effects of unloading conditions on the trabecular bone morphology? In an attempt to answer this question, rat tail-suspension was chosen as a model that mimics the microgravity environment. Over 23 days, histomorphometric analysis was carried out on cross-sections of tibias of the suspended animals. The slices were digitalized and images discretized to obtain osteocyte distribution and apparent bone density. Based on these experimental data, finite element simulations were conducted to evaluate the bone loss and the change in trabecular architecture similar to those observed after a spaceflight. The numerical model is driven by a remodeling law that takes into account the nonuniform osteocyte distribution that may itself provide mechanoreception. We used the bone density rate of change from the remodeling theory and a time stepping algorithm witch are implemented in a finite element software. This approach takes into account the unloading effects on bone remodeling process and permits to confront experimental and numerical data. We showed that there is a good agreement between these data, particularly at the beginning of the simulated bone mass loss during the rat tail-suspension experiment. Indeed, we obtained a variation of 5.25% at day 7 (D7), 2.09% at day 13 (D13) and finally, 51.03% at day 23 (D23). Despite that last variation, the proposed theoretical model can be suitable to simulate the alteration of bone mineral density under the specific unloading conditions of the rat tail-suspension model.

Sensitivity of alveolar macrophages to substrate mechanical and adhesive properties.

In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on substrates of increasing Young's modulus: (i) a monolayer of alveolar epithelial cells representing the supple (approximately 0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high intermediate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD-coated beads and corrected for actual bead immersion). Macrophage shape dramatically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligible effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized numerical model describing how low cell height enhances the substrate-stiffness-dependence of the apparent (measured) AM stiffness. Altogether, these results suggest that macrophages are able to probe their physical environment but the mechanosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity.