Claudia Siverino

Orthopedic device‐related infection (ODRI) is a devastating complication, often requiring repeated revision surgeries and prolonged antibiotic treatment. Injectable biodegradable biomaterials that deliver and maintain high local concentrations of antibiotics to the surgical field may eliminate the need for additional surgeries while improving treatment outcomes. In this study, we introduce a Gel for Delivery of Antibiotics (GEDAI) for local administration of tobramycin (T), gentamicin (G) or gentamicin-vancomycin (GV). GEDAI was developed with translatability and ease of intraoperative handling as design goals. GEDAI displayed storage and loss moduli, shear thinning, and elastic recovery properties appropriate for intraoperative applications, with good adhesion to stainless steel and tissues but reduced adhesion to surgical gloves. In vitro assays showed no adverse effects of the GEDAI on viability of fibroblasts, macrophages and endothelial cells, or osteogenic differentiation of human bone marrow stromal cells (hBMSCs). Methicillin susceptible and resistant Staphylococcus aureus (MSSA and MRSA), growing in planktonic culture or biofilm, were significantly reduced after exposure to the antibiotic loaded-GEDAI compared to GEDAI without antibiotics (p < 0.05). In three distinct in vivo studies in sheep, GEDAI-T reached a peak local concentration above 2000 μg/mL within 2 h and substantially reduced bacterial counts in two models with implant retention (GEDAI-T or GEDAI-G), and completely eradicated infection in an implant exchange model (GEDA-GV). GEDAI directly targets two unmet needs in ODRI: achieving bactericidal local concentrations without systemic toxicity and providing a ready-to-use formulation. The antibiotic-loaded GEDAI formulations promise to improve treatment outcomes in challenging infection cases, potentially reducing healthcare costs and patient burden in postoperative infection management.

Aims: Chronic bone and joint infections caused by Staphylococcus epidermidis can be difficult to treat and are prone to recurrence. In this study, established human osteoblast- and osteocyte-like cell line models were used in vitro to investigate the intracellular persistence of S. epidermidis as a potential mechanism for infection persistence. Methods: The SaOS2 human osteosarcoma cell line was differentiated into osteoblast-like (SaOS2-OB) and osteocyte-like (SaOS2-OY) stages and infected with four S. epidermidis strains at varying multiplicities of infection. Susceptibilities to lysostaphin, a known bacteriolytic enzyme, and a panel of antibiotics were determined to identify an antimicrobial capable of clearing extracellular bacteria while preserving intracellular bacterial viability. Bacterial survival and number were quantified by colony-forming unit and droplet digital polymerase chain reaction (PCR), while host gene expression was analyzed by real-time PCR. Results: Lysostaphin was ineffective against all four S. epidermidis strains, while 10 × minimal bactericidal concentration levofloxacin consistently eliminated extracellular but not intracellular bacteria. Intracellular S. epidermidis at multiplicities of infection of 1,000 persisted in SaOS2-OY for 14 days at 10 × minimal bactericidal concentration levofloxacin, while it was cleared within five days in SaOS2-OB cells. Droplet digital PCR genome copy analysis identified a higher number of bacteria than conventional culture, indicating more accurate counting and a transition of the intracellular bacteria to a non-culturable state. Proinflammatory and bone remodelling responses by both bone cell types were evident upon infection, with increased messenger RNA levels of CCL5 , CXCL6 , CXCL10 , SOST , receptor activator of nuclear factor kappa-B ligand ( RANKL ), and matrix metalloproteinase 13 ( MMP13 ). Conclusion: We report the first model of intracellular S. epidermidis persistence in osteocyte-like cells, which provides a robust platform for mechanistic studies of chronic bone and joint infections and the preclinical screening of novel therapeutics targeting intracellular bacteria. Cite this article: Bone Joint Res 2026;15(8):987–997.

Critical-sized long-bone defects and non-unions are among the most challenging complications in fracture care, as they lack the capacity for spontaneous he

Background: Dexamethasone (DEX) is used in vitro to promote osteogenic differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). In clinical use, however, glucocorticoids induce osteoblast and osteocyte apoptosis while increasing osteoclast survival, leading overall to osteoporosis and high fracture risk. The overall impact of DEX on the differentiation of human progenitor cells remains contradictory and not fully understood, highlighting the need for further investigation using sequencing approaches as in vitro results will naturally influence further translational research. Methods: hBMSCs were induced to osteogenic differentiation for 7 days using different concentrations of either DEX or the nonsteroidal glucocorticoid receptor agonist (+)-ZK216348. cDNA library preparation and RNA sequencing (RNAseq) were performed using Oxford Nanopore Technologies. Differentially expressed genes and pathways associated to the transactivation or transrepression activity of DEX were identified. Sequencing results were validated by qPCR, protein analysis, and with a functional assay on peripheral blood mononuclear cells to determine the overall effect of the BMSC supernatant. Results: Hierarchical clustering of RNAseq data identified eight subclusters with shared regulatory patterns. Enrichment analysis revealed that both upregulated and downregulated genes are involved in ossification and extracellular matrix organization pathways. Several pro- and anti-inflammatory genes were differentially regulated. qPCR analysis validated the upregulation of CXCL1 , CXCL8 , IL18 , and COL8A1 , while MMP1 and CXCL12 expression decreased in response to DEX. Comparing DEX results with those obtained using (+)-ZK216348 helped distinguish the potential mechanisms regulating the expression of specific genes. Notably, CXCL8 upregulation occurred through transactivation, whereas COL8A1 upregulation is downstream of a transrepressed gene. Further in vitro experiments confirmed that DEX significantly increased CXCL8 expression and IL-8 secretion. However, hPBMC responses indicated no significant pro- or anti-inflammatory effects from hBMSC conditioned medium. Conclusions: In conclusion, the effects of DEX on the transcriptome of hBMSCs in a pro-osteogenic environment do not fully replicate the acquisition of an osteogenic phenotype. Several genes associated with ossification, extracellular matrix organization, and inflammation were dysregulated. The unique expression patterns of pro-inflammatory cytokines and collagen types warrant further investigation to elucidate their roles in osteogenic differentiation and bone homeostasis.

Fracture-related infection (FRI) remains one of the most significant complications in the surgical treatment of fractured bones. The risk of developing an infection associated with any implanted device is increased by the presence of the device itself, which acts as a foreign body, providing a surface for adhesion and biofilm formation to any microorganism entering the wound.The biomechanical properties of fracture fixation devices play a crucial role in the healing process of surgically treated fractures. It is well established that the biomechanics involved in fracture fixation significantly influence healing outcomes. Additionally, the stability of the fixation is considered a critical factor in the development of FRI.Several preclinical studies provide evidence supporting the link between stable fixation and a reduced risk of infection. In a mouse model of plating osteosynthesis with stable and rigid MouseFix fixation infected with Staphylococcus epidermidis, it was observed that infection rates were influenced by fracture stability. However, no differences were detected when using S. aureus, which caused a more severe infection, resulting in osteolysis and loss of stability in both groups. Additionally, biomechanical instability led to a stronger local inflammatory response, characterized by increased levels of inflammatory markers such as granulocyte colony-stimulating factor (G-CSF), keratinocyte chemoattractant (KC), and interleukin (IL)-6 within the bone.Lately the use of the implant load sensor (fracture monitor) in a sheep model of S. aureus induced FRI revealed the potential of measuring relative implant load (RIL) as a surrogate for fracture healing. The use of sensors for measuring implant load as an objective and quantitative indicator of fracture stability can provide valuable information on the progression of healing. In the sheep study, the fracture monitor was able to track the implant load as the infection was induced and treated. Implant load appeared to correspond to the degree of infection burden, with high correlation values between the bacterial load at revision and euthanasia and the sensor data. The implant load could identify infected animals by measuring the highest RIL values in culture-positive animals at the end of the study and by identifying delayed healing associated with infection.Stability plays a crucial yet not fully understood role in managing FRI. The continued development and application of mechanical load sensors, and the ever-improving control over biomechanical conditions in preclinical fracture models promises significant advances in our understanding. How this may impact patient care remains to be determined, but deeper insights are gradually revealing the connection between biomechanics, immunology and bone healing in infected and non-infected scenarios.

Musculoskeletal infections (MSKIs) pose significant challenges in orthopedic surgery. These infections are often caused by Staphylococcus aureus and S. epidermidis. Early diagnosis is crucial, as it allows prompt treatment before the infection becomes chronic and more difficult to manage. This study evaluated the expression of microRNAs (miRNAs) in the serum of patients with staphylococcal MSKI and their potential as biomarkers. Serum samples from patients with confirmed staphylococcal MSKI were used for RNA sequencing and validation (n = 24 S. aureus, n = 25 S. epidermidis and n = 25 controls). In S. aureus‐infected patients compared to uninfected controls, 23 miRNAs were upregulated and 20 downregulated, while 94 miRNAs were upregulated and 31 downregulated in those infected with S. epidermidis. Validation experiments revealed the upregulation of hsa‐miR‐1246, hsa‐miR‐483‐5p, and hsa‐miR‐1290, and the downregulation of hsa‐miR‐1‐3p, hsa‐miR‐148b‐3p, and hsa‐miR‐23a‐3p in S. aureus‐infected patients compared to both S. epidermidis‐infected patients and uninfected controls. Human peripheral blood mononuclear cells treated in vitro with bacterial culture supernatants exhibited a similar pattern of miRNA expression. Receiver operating characteristic (ROC) curve analyses demonstrated that combining four miRNAs (miR‐1246, miR‐1290, miR‐148b‐3p, and miR‐23a‐3p) achieved a high diagnostic accuracy, with an area under the curve of 0.96, distinguishing S. aureus from S. epidermidis‐infected patients. In summary, this study identified four differentially expressed miRNAs in S. aureus‐infected patients compared to those with S. epidermidis infections and uninfected controls. These findings allow miRNA to be used as MSKI markers and to study their effect on immune responses during bacterial infections.

Treatment of orthopedic device‐related infection (ORDI) generally requires a combination of medical and surgical interventions for successful treatment outcomes. Many cases are treated with a two‐stage revision, whereby contaminated implants and necrotic tissues are removed, and dead space is managed with a temporary, non‐resorbable polymethyl methacrylate (PMMA) spacer loaded with antibiotics. Weeks later, this is replaced with a bone graft or similar material to aid bone healing. However, this two‐stage approach is quite a burden for the patient, and infection may still recur. The use of a 3D‐printed, absorbable, antibiotic‐releasing material that also promotes bone healing would be a promising alternative that produces the exact geometry of the missing bone and eliminates the need for a second surgery. In this study, we investigated whether a novel 3D‐printed, antibiotic‐loaded, osteoconductive calcium phosphate scaffold (CPS) is effective in the single‐stage revision of an infected segmental bone defect model in rabbits. A 5‐mm segmental defect of the radius of female New Zealand White rabbits (n = 64), stabilized with cerclage wire, was inoculated with Staphylococcus aureus. After 4 weeks, the infected bone fragment was removed, the site debrided, and the bone defect was either left empty (Control group) or filled with a PMMA spacer with gentamicin, CPS loaded with rifampicin or non‐loaded CPS. The animals were also managed with systemic cefazolin for 4 weeks. An additional group received vancomycin‐loaded CPS without adjunctive systemic antibiotic therapy. All animals were euthanized 8 weeks after revision and assessed by quantitative bacteriology or semiquantitative histopathology. The antibiotic‐loaded scaffolds (PMMA‐Genta and CPS‐Rif) in the animals receiving systemic antibiotic treatment resulted in a reduction in bacterial count at euthanasia compared to controls (rabbits receiving systemic antibiotic alone and in which the defect was left empty). The PMMA‐Genta induced a significant CFU reduction (p = 0.0486) compared to controls. The infection rate was also reduced from 80% in the control group to 50% for the groups receiving local and systemic antibiotics. The CPS‐Vanco group for local delivery without adjunctive systemic antibiotic therapy resulted in a lower infection rate, but the CFUs in these samples at euthanasia were comparable with those of the control group. The findings show that treating an ODRI with PMMA‐Genta yields the best results for infection eradication; however, it does not provide the reconstruction opportunity that the antibiotic‐loaded CPS does. Even though it is not comparable to the PMMA‐Genta, the antibiotic‐loaded CPS showed a reduction in infection rates. The use of local antibiotics alone is insufficient to eradicate the infection.

Introduction: Bone and joint infection (BJI) is often characterized by severe inflammation and progressive bone destruction. Osteocytes are the most numerous and long-lived bone cell type, and therefore represent a potentially important long-term reservoir of bacterial infection. Staphylococcus aureus is known to establish stable intracellular osteocytic infections, however, little is known about the less virulent yet second most prevalent BJI pathogen, S. epidermidis, associated with late-diagnosed, chronic BJI. Thus, this study sought to establish an in vitro model to study the infection characteristics of S. epidermidis in human osteocyte-like cells.Methods: SaOS2 cells (1 ×10⁴ cells/cm²) were grown to confluence either without differentiation, representing an osteoblast-like (OB) state (SaOS2-OB) or differentiated to an osteocyte-like stage (SaOS2-OY), using established methods. Four S. epidermidis strains used (ATCC-12228, ATCC-14990, ATCC-35984 and a clinical osteomyelitis strain RAH-SE1) were tested to be Lysostaphin-resistant, necessitating antibiotic (Levofloxacin) control of extracellular bacteria. Infection of host cells (OB or OY) was tested at three multiplicities of infection (MOI: 10, 100 and 1000). Extracellular bacteria were controlled by overnight incubation at a 10X minimum inhibitory concentration (MIC) of Levofloxacin and thereafter at 1XMIC. At each time point (days 1, 3, 5) viable intra- and extracellular bacteria were quantified.Result: All strains displayed similar intracellular infection and persistence capabilities in SaOS2-OB and SaOS2-OY. Independent of MOI, intracellular bacteria in SaOS2-OB decreased over time, becoming non-culturable by day 5. In contrast, SaOs2-OY displayed enhanced intracellular bacterial persistence at each time point. In the presence of increased Levofloxacin concentration (10XMIC), S. epidermidis could persist intracellularly for at least 14 days.Conclusion: This study showed for the first time that S. epidermidis can infect human osteocytes and persist intracellularly. Additionally, even a 10xMIC antibiotic concentration failed to eradicate intracellular bacteria, suggesting that persistence within osteocytes could contribute to treatment failure and establishment of chronic BJI.

Staphylococcus aureus has multiple mechanisms to evade the host's immune system and antibiotic treatment. One such mechanism is the invasion of the osteocyte lacuno‐canalicular network (OLCN), which may be particularly important in recurrence of infection after debridement and antibiotic therapy. The aim of this study was to develop an ex vivo model to facilitate further study of S. aureus invasion of the OLCN and early‐stage testing of antibacterial strategies against bacteria in this niche. The diameter of the canaliculi of non‐infected human, sheep, and mouse bones was measured microscopically on Schmorl's picrothionin stained sections, showing a large overlap in canalicular diameter. S. aureus successfully invaded the OLCN in all species in vitro as revealed by presence in osteocyte lacunae in Brown and Brenn‐stained sections and by scanning electron microscopy. Murine bones were then selected for further experiments, and titanium pins with either a wild‐type or ΔPBP4 mutant S. aureus USA300 were placed trans‐cortically and incubated for 2 weeks in tryptic soy broth. Wild‐type S. aureus readily invaded the osteocyte lacunae in mouse bones while the ΔPBP4 showed a significantly lower invasion of the OLCN (p = 0.0005). Bone specimens were then treated with gentamicin, sitafloxacin, R14 bacteriophages, or left untreated. Gentamicin (p = 0.0027) and sitafloxacin (p = 0.0280) significantly reduced the proportion of S. aureus‐occupied lacunae, whilst bacteriophage treatment had no effect. This study shows that S. aureus is able to invade the OLCN in an ex vivo model. This ex vivo model can be used for future early‐stage studies before proceeding to in vivo studies.

With the introduction and continuous improvement in operative fracture fixation, even the most severe bone fractures can be treated with a high rate of successful healing. However, healing complications can occur and when healing fails over prolonged time, the outcome is termed a fracture nonunion. Nonunion is generally believed to develop due to inadequate fixation, underlying host-related factors or infection. Despite the advancements in fracture fixation and infection management, there is still a clear need for earlier diagnosis, improved prediction of healing outcomes and innovation in the treatment of nonunion. This review provides a detailed description of nonunion from a clinical perspective including state of the art in diagnosis, treatment, and currently available biomaterials and orthobiologics.Subsequently, recent translational development from the biological, mechanical and infection research fields are presented including the latest in smart implants, osteoinductive materials and in silico modeling. The first challenge for future innovations is to refine and to identify new clinical factors for the proper definition, diagnosis and treatment of nonunion. However, integration of in vitro, in vivo, and in silico research will enable a comprehensive understanding of nonunion causes and correlations, leading to the development of more effective treatments.