Hyperglycemia, a hallmark of diabetes, can induce inflammatory programming of macrophages. The macrophage scavenger receptor CD163 internalizes and degrades hemoglobin-haptoglobin (Hb-Hp) complexes built due to intravascular hemolysis. Clinical studies have demonstrated a correlation between impaired scavenging of Hb-Hp complexes via CD163 and diabetic vascular complications. Our aim was to identify whether hyperglycemia is able to amplify inflammation via Hb-Hp complex interactions with the immune system. M(IFNγ), M(IL-4), and control M0 macrophages were differentiated out of primary human monocytes in normo- (5 mM) and hyperglycemic (25 mM) conditions. CD163 gene expression was decreased 5.53 times in M(IFNγ) with a further decrease of 1.99 times in hyperglycemia. Hyperglycemia suppressed CD163 surface expression in M(IFNγ) (1.43 times). Flow cytometry demonstrated no impairment of Hb-Hp uptake in hyperglycemia. However, hyperglycemia induced an inflammatory response of M(IFNγ) to Hb-Hp1-1 and Hb-Hp2-2 uptake with different dynamics. Hb-Hp1-1 uptake stimulated IL-6 release (3.03 times) after 6 h but suppressed secretion (5.78 times) after 24 h. Contrarily, Hb-Hp2-2 uptake did not affect IL-6 release after 6h but increased secretion after 24 h (3.06 times). Our data show that hyperglycemia induces an inflammatory response of innate immune cells to Hb-Hp1-1 and Hb-Hp2-2 uptake, converting the silent Hb-Hp complex clearance that prevents vascular damage into an inflammatory process, hereby increasing the susceptibility of diabetic patients to vascular complications.

The impact of microbial biofilm growth poses a threat to both human health and the performance of industrial systems, manifesting as a global crisis with noteworthy economic implications for modern society. Exploring new methods and alternative approaches for the detection of biofilm signatures are imperative for developing optimized and cost-effective strategies that can help to identify early-stage biofilm formation. Clinical diagnostic technologies are constantly looking for more affordable, practical and faster methods of prevention and detection of chronic infections in periprosthetic joint infections (PJIs), which are often characterized by biofilm formation on implant surfaces. Staphylococcus epidermidis (SE) is especially known for its strong biofilm production and is considered a leading cause of biomaterial-associated infections, including PJIs. Implant-associated infections are severe and difficult to treat, therefore it is crucial to continue identifying bacterial biomarkers that contribute to its structural stability and attachment to implant surfaces. This study presents a pioneering approach for fast spectral detection of biofilm formation with a novel mid-infrared (MIR) scanning system. To highlight the advantages of our MIR system, we performed a comparative analysis with measurements from a commercially available Fourier-transform infrared (FTIR) scanner. We have assessed SE biofilms grown for 3 days comparing the processing times between a commercially available infrared (IR) scanning system (∼8 h/cm²), and our innovative scanning approach with rapid self-built MIR detection, achieving a reduction in scanning time to seconds. K-means clustering analysis identified pronounced differences in distribution of clusters, representing a significant variation between biofilm producing (RP62A) and non-biofilm producing (ATCC 12228) bacterial strains. The distribution serves as a critical tool for identifying biofilm phenotypes, particularly where poly-N-acetylglucosamine (PNAG), a key constituent of extracellular polymeric substances (EPS) in S. epidermidis, represents the dominant mass fraction in the samples analyzed by our infrared (IR) scanning systems. In addition to faster processing times, our novel MIR system demonstrated significantly higher sensitivity compared to FTIR, enabling clear differentiation between the chemical signatures of biofilm and planktonic strains. The corresponding novel approach integrates advanced data analytics with a newly designed rapid MIR prototype, enabling optimized and swift detection of biofilm signatures. These signatures, now recognized as critical targets in diagnosing complex infections, provide an alternative to traditional microbial detection methods in clinical diagnostics.

Background/Objectives: Staphylococcus epidermidis (RP62A) and Staphylococcus aureus (UAMS-1) are clinically relevant pathogens frequently implicated in implant-associated infections due to their ability to form biofilms. RP62A is typically linked to persistent, chronic, low-grade infections, whereas UAMS-1 is associated with acute, invasive disease. Both strains serve as representative models for chronic and acute periprosthetic joint infections (PJIs). The objective of this study was to examine and compare in vitro biofilm formation by RP62A and UAMS-1 on orthopaedic materials/disc surfaces of defined composition. Methods: In vitro biofilm formation assays were performed using orthopaedic disc surfaces composed of cobalt–chromium alloy (CoCr), titanium alloy (Ti), and polyethylene (PE) after 72 h of incubation. Biofilm biomass was quantified using crystal violet staining, with absorbance measured at OD₅₇₀. A polystyrene (PS) surface served as a control. Additionally, retrieved orthopaedic explant components were used as substrates for in vitro biofilm assays, in which RP62A was incubated for 72 h on the explanted surfaces. Supporting assays on glass slides were conducted to examine strain-specific biofilm-related architecture. Results: In vitro biofilm mass quantification assays showed strong biofilm formation by RP62A across all tested surfaces, with the highest absorbance on CoCr (OD₅₇₀ = 5.80 ± 0.19). Notably, biofilm formation on CoCr was 76% higher compared to PS (p < 0.0001). No significant differences were observed among all three surface discs (p > 0.1). Biofilm formation was highest on PE for UAMS-1 (OD₅₇₀ = 1.29 ± 0.09) and was significantly greater than on Ti (178%, p < 0.001) and CoCr (196%, p < 0.0001). In the in vitro assays performed on retrieved explant components, RP62A showed pronounced biofilm accumulation on polyethylene tibial inserts, particularly in regions of mechanical wear and friction. Supporting assays on glass slides were performed to examine strain-specific surface microstructural, revealing dense network-like structures for RP62A and thinner, discontinuous layers for UAMS-1. Conclusions: RP62A formed dense biofilms in vitro on multiple orthopaedic implant materials and retrieved explant components, consistent with its association with chronic periprosthetic joint infections. Increased biofilm accumulation was observed on mechanically worn polyethylene surfaces. In contrast, UAMS-1 showed lower biofilm formation on metallic disc surfaces, indicating strain- and material-dependent differences. These findings highlight the relevance of implant material selection and surface integrity for strategies targeting biofilm-associated implant infections.

Titanium is widely used for orthopedic and dental implants because of its biocompatibility and mechanical properties. However, corrosion and mechanical wear can result in the release of titanium-containing particulate debris, which may contribute to chronic peri-implant inflammation and implant failure. Macrophages are primary innate immune cells that recognize and respond to implant-derived particles, yet the effects of TiO ₂ particles on the macrophage-derived chitinase family members chitotriosidase (CHIT1) and YKL-40 (CHI3L1) remain poorly understood. Here, we investigated the effects of TiO ₂ particles derived from a 15-nm rutile nanoparticulate starting material that exhibited substantial and preparation-dependent agglomeration during laser diffraction analysis on primary human M0, M1, and M2 macrophages. Metabolic activity, assessed using the Alamar Blue assay, was maintained or increased following exposure to 25, 50, 100, and 200 ppm TiO ₂ , with a statistically significant increase observed at 100 ppm in M0 macrophages, while no significant changes were detected in M1 or M2 macrophages. Transcriptomic analysis identified CHIT1 and CHI3L1 as the two most upregulated members of the Glyco_18 protein family. RT-qPCR confirmed significant induction of CHIT1 following TiO ₂ exposure in M0, M1, and M2 macrophages, while CHI3L1 expression was significantly increased in M0 and M2 macrophages. Increased gene expression was accompanied by a concentration-dependent increase in YKL-40 secretion, with statistically significant increases observed from 100 ppm TiO ₂ onwards. At 100 ppm, extracellular CHIT1 protein levels increased significantly in M0 and M1 macrophages, despite no corresponding significant increase in CHIT1 enzymatic activity. Immunofluorescence analysis revealed altered intracellular localization of CHIT1 following TiO ₂ exposure, with accumulation in the trans-Golgi network (TGN) of M1 macrophages and disruption of TGN organization in M0 and M2 macrophages. Furthermore, CHIT1 and YKL-40 were detected in CD68-positive macrophages within peri-implant tissues obtained from patients undergoing removal of titanium fracture fixation implants, supporting the clinical relevance of the in vitro findings. Collectively, these findings identify CHIT1 and YKL-40 as macrophage-associated proteins responsive to TiO ₂ particle exposure and support their further investigation in biomaterial-associated inflammation.