Abstract. Background. The aim of the present experimental study was to analyse vancomycin elution kinetics of nine bone fillers used in orthopaedic and trauma surgery over 42 consecutive days. Methods. Two allograft bone chips (carriers 1 and 2), a calcium-sulfate matrix (carrier 3), a hydroxyapatite/calcium-sulphate composite (carrier 4), four bone cements (carriers 5-8) and a pure tricalcium phosphate matrix (carrier 9), either already contained vancomycin, or were mixed with it following manufacturer's recommendations. Over 42 days, half of elution medium was substituted by the same amount of PBS at 9 distinct time points. Vancomycin concentration in obtained samples were measured with a kinetic microparticle immunoassay, and masses consecutively calculated. To enhance comparability between carriers analysed, vancomycin mass released related to overall mass within each probe was determined. Notably, elution kinetics of carriers 1 to 4 have been published previously. Results. All carriers initially released high vancomycin masses, followed by constant reduction later into the experiment. Mean initial vancomycin masses released after 4 hours were highest for carriers 1 (337.7 ± 76.2 mg), 9 (68.4 ± 4.9 mg), and 2 (49.0 ± 54.6 mg). From prefinal (35 days) to last measurement (42 days) carriers 2 (8.6 ± 4.8 mg), 1 (2.4 ± 1.0 mg), and 5 (0.1 ± 0.1 mg) had released highest vancomycin masses. Notably, all five bone cements tested only released a small percental amount of their total mass up to the last measurement (42 days; 2.1% – 9.3%), whilst allografts and resorbable synthetic bone fillers discarded high percental values (22.5% – 79.2%). Conclusions. Elution kinetics differ between 9 antibiotic-loaded bone fillers, with high vancomycin masses released by allografts and resorbable bone fillers over time. Transferred to clinical practice, these may be favoured over bone cements in case prolonged and high antibiotic release is warranted rather than mechanical stability

Introduction. Various biomaterials and bone graft substitute technologies for use in osteomyelitis treatment are currently used in clinal practice. They vary in mode of action (with or without antibiotics) and clinical application (one-stage or two-stage surgery). This systematic review aims to compare the clinical evidence of different synthetic antimicrobial bone graft substitutes and antibiotic-loaded carriers in eradicating infection and clinical outcome in patients with chronic osteomyelitis. Methods. Systematic review according to PRISMA statement on publications 2002-2023. MESH terms: osteomyelitis and bone substitutes. FREE terms: chronic osteomyelitis, bone infection. A standardized data extraction form was be used to extract data from the included papers. Results. Publications with increased methodological quality and clinical evidence for biomaterials in osteomyelitis treatment were published in the last decades. High 85-95% eradication rates of osteomyelitis were observed for various resorbable Ca-P and/or Ca-S biomaterials combined with antibiotics and S53P4 bioactive glass. Level of evidence varies significantly between products. Antibiotic pharmacokinetic release profiles vary between resorbable Ca-P and/or Ca-S biomaterials. Conclusion. Given the high 85-95% eradication rates of osteomyelitis by various resorbable Ca-P and/or Ca-S biomaterials combined with antibiotics and S53P4 bioactive glass, one-stage treatment is preferred. Surgeons should be aware of variations in mechanical properties and antibiotic pharmacokinetic release profiles between Ca-P and CA-s products. Mechanical, biological and antimicrobial properties of bioactive glass are formulation dependent. Currently, only S53P4 bioactive glass has proven antimicrobial properties. Based on this systematic review antibiotic loaded fleeces should be used with caution and restraint

Aim. There is controversy regarding the use of Antibiotic-loaded cement (ALBC) as compared to non-antibiotic-loaded cement (NALBC) to reduce the overall infection rate without affecting implant survival or adding additional risks on fixation for primary total knee arthroplasty (TKA). Method. To conduct the analysis, we utilized the Catalan Arthroplasty Registry (RACat) for the TKAs implanted between 2005 and 2017. The primary variable recorded was the use of cement with or without antibiotics. Other recorded variables included were age, sex, diabetes mellitus, obesity, Charlson index and type of hospital. We analyzed the effect of ALBC vs. NALBC in reducing the risk of prosthetic infection at 3, 6, 12, and 24 months as well as prosthetic survival due to mechanical causes at 1, 5, and 10 years. Univariate and multivariable analyses of risk factors were conducted. Thereby, an interactive predictive model that determines the risk of prosthetic infection based on each patient's characteristics was created. Results. A total of 28,287 TKAs from the RACat were analyzed. In that total, there were 19,788 NALBC and 8,499 ALBC. The infection rates for TKAs with NALBC vs. those with ALBC at 3, 6, 12, and 24 months were respectively: 1.69% vs. 1.39% (p=0.132); 1.81% vs. 1.56% (p=0.147); 2.14% vs. 1.73% (p=0.030); 2.51% vs. 1.86% (p=0.001). A statistically significant reduction in periprosthetic infection rate was observed in the ALBC group at 12 and 24 months. No differences were observed between the two groups in terms of prosthetic mechanical survival. Being younger, male and having had previous knee surgery or having a high comorbidity index all led to a higher risk of prosthetic revision due to infection. Conclusions. The use of ALBC as a fixation method for TKA leads to a reduction in the risk of prosthetic infection without altering the mechanical survival of the implant. The creation of a predictive model helps determine the individualized risk of prosthetic infection based on the patient's characteristics

Introduction. Intervertebral disc degeneration has been associated with low back pain (LBP) which is a major cause of long-term disability worldwide. Observed mechanical and biological modifications have been related to decreased water content. Clinical traction protocols as part of LBP management have shown positive outcomes. However, the underlying mechanical and biological processes are still unknown. The study purpose was to evaluate the impact of unloading through traction on the mechanobiology of healthy bovine tail discs in culture. Method. We loaded bovine tail discs (n=3/group) 2h/day at 0.2Hz for 3 days, either in dynamic compression (-0.01MPa to -0.2MPa) or in dynamic traction (-0.01MPa to 0.024MPa). In between the dynamic loading sessions, we subjected the discs to static compression loading (-0.048MPa). We assessed biomechanical and biological parameters. Result. Over the 3 days of loading, disc height decreased upon dynamic compression loading but increased upon unloading. The neutral zone was restored for all samples at the end of the dynamic unloading. Upon dynamic compression, the stiffness increased over time while the hysteresis decreased. Upon dynamic unloading, sulfated glycosaminoglycan (sGAG) release in the medium was lower at the endpoint. In the outer annulus fibrosus (AFo), we saw a higher water/sGAG of at least 30%. In the nucleus pulposus, COL2 mRNA was expressed more highly upon dynamic unloading while MMP3, iNOS and TRPV4 expression levels were lower. In the AFo of the unloading group, COL2 expression was higher but COL1 was lower. Conclusion. The biomechanical and biological results consistently indicate that dynamic unloading of healthy bovine discs in culture facilitates water uptake and promotes an anti-catabolic response which reflects a function optimization of the disc. This work combines biomechanical and biological results and opens the door to evidence-based improvement of regenerative protocols for degenerated discs and conservative LBP management. This study is funded by AO Foundation and AO Spine

Introduction. Promoting bone mass homeostasis keeps skeleton away from osteoporosis. a-Ketoglutarate (a-KG) is an indispensable intermediate of tricarboxylic acid cycle (TCA) process for cellular energy production. a-KG mitigates cellular senescence, tissue degeneration, and oxidative stress. We investigated whether a-KG affected osteoblast activity or osteoporosis development. Method. Serum and bone specimens were biopsied from 26 patients with osteoporosis or 24 patients without osteoporosis who required spinal surgery. Ovariectomized or aged mice were fed 0.25% or 0.75% a-KG in drinking water for 8 – 12 weeks ad libitum. Bone mineral density, trabecular/cortical bone microarchitecture, mechanical strength, bone formation, and osteoclastic erosion were investigated using mCT, material testing device, in vivo calcein labelling, and TRAP histochemical staining. Serum a-KG, osteocalcin, and TRAP5b levels were quantified using ELISA kits. Bone-marrow mesenchymal cells and macrophages were incubated osteogenic and osteoclastogenic media. Histone H3K27me3 levels and enrichment were investigated using immunoblotting and chromatin precipitation-PCR. Result. Serum a-KG levels in patients with osteoporosis were less than controls; and were correlated with T-scores of hips (R2 = 0.6471, P < 0.0001) and lumbar spine (R2 = 0.7235, P < 0.001) in osteoporosis (AUC = 0.9941, P < 0.001). a-KG supplement compromised a plethora of osteoporosis signs in ovariectomized or aged mice, including bone mass loss, trabecular bone microarchitecture deterioration, and mechanical strength loss. It elevated serum osteocalcin levels and decreased serum TRAP5b. a-KG preserved caclein-labelling bone formation and repressed osteoclast resorption. It reversed osteogenic differentiation of bone-marrow stromal cells and reduced osteoclast formation in ovariectomized mice. Mechanically, a-KG attenuated H3K27 hypermethylation and Runx2 transcription repression, improving mineralized matrix production in osteogenic cells. Conclusion. Decreased serum a-KG is correlated with human and murine osteoporosis. a-KG reverses bone loss by repressing histone methylation in osteoblasts. This study highlighted a-KG supplement as a new biochemical option for protecting osteoporosis

Introduction. This study aimed to evaluate the effectiveness of a novel intraoperative navigation platform for total knee arthroplasty (TKA) in restoring native knee joint kinematics and strains in the medial collateral ligament (MCL) and lateral collateral ligament (LCL) during squatting motions. Method. Six cadaver lower limbs underwent computed tomography scans to design patient-specific guides. Using these scans, bony landmarks and virtual single-line collateral ligaments were identified to provide intraoperative real-time feedback, aided in bone resection, implant alignment, tibiofemoral kinematics, and collateral ligament elongations, using the navigation platform. The specimens were subjected to squatting (35°-100°) motions on a physiological ex vivo knee simulator, maintaining a constant 110N vertical ankle load regulated by active quadriceps and bilateral hamstring actuators. Subsequently, each knee underwent a medially-stabilized TKA using the mechanical alignment technique, followed by a retest under the same conditions used preoperatively. Using a dedicated wand, MCL and LCL insertions—anterior, middle, and posterior bundles—were identified in relation to bone-pin markers. The knee kinematics and collateral ligament strains were analyzed from 3D marker trajectories captured by a six-camera optical system. Result. Both native and TKA conditions demonstrated similar patterns in tibial valgus orientation (Root Mean Square Error (RMSE=1.7°), patellar flexion (RMSE=1.2°), abduction (RMSE=0.5°), and rotation (RMSE=0.4°) during squatting (p>0.13). However, a significant difference was found in tibial internal rotation between 35° and 61° (p<0.045, RMSE=3.3°). MCL strains in anterior (RMSE=1.5%), middle (RMSE=0.8%), and posterior (RMSE=0.8%) bundles closely matched in both conditions, showing no statistical differences (p>0.05). Conversely, LCL strain across all bundles (RMSE<4.6%) exhibited significant differences from mid to deep flexion (p<0.048). Conclusion. The novel intraoperative navigation platform not only aims to achieve planned knee alignment but also assists in restoring native knee kinematics and collateral ligament behavior through real-time feedback. Acknowledgment. This study was funded by Medacta International (Castel San Pietro, Switzerland)

Introduction. Tendon ruptures represent one of the most common acute tendon injuries in adults worldwide, affecting millions of people anually and becoming more prevalent due to longer life expectancies and sports activities. Current clinical treatments for full tears are unable to completely restore the torn tendons to their native composition, structure and mechanical properties. To address this clinical challenge, tissue-engineered substitutes will be developed to serve as functional replacements for total tendon ruptures that closely resemble the original tissue, restoring functionality. Method. Water borne polyurethanes (WBPU) containing acrylate groups, specifically polyethylene glycol methacrylate (PEGMA) or 2-hydroxyethyl methacrylate (HEMA), were combined with mouse mesenchymal stem cells (MoMSCs) and heparin sodium to formulate bioinks for the fabrication of scaffolds via extrusion-based 3D bioprinting. Result. The biocompatibility of acrylated-WBPUs was confirmed in 2D with MoMSCs using lactate dehydrogenase assay, DNA assay and live/dead assays. Cell-laden scaffolds were 3D-bioprinted by encapsulating MoMSCs at varying cell densities within the acrylated WBPUs. The resulting 3D structures support cell viability and proliferation within the scaffolds, as confirmed by live/dead assay, lactate dehydrogenase assay and DNA assays. Differentiation studies in the 3D-bioprinted scaffolds demonstrated the phenotype transition of MoMSCs toward tenocytes through gene expression and protein deposition analysis. The inclusion of sodium heparin in the bioinks revealed increased synthesis of matrix assembly proteins within the 3D-bioprinted constructs. Conclusion. The developed bioinks were biocompatible and printable, supporting cell viability within the 3D-bioprinted scaffold. The fabricated cell-laden constructs sustained cell proliferation, differentiation, and tissue formation. The addition of heparin sodium enhanced tissue formation and organization, showing promising results for the regeneration of tendon total ruptures. Principio del formularioThis work was supported by the Spanish State Research Agency (AEI) under grant No CPP2021-008754. The authors would like to thank their partners in the project, which are in charge of the synthesis of heparin sodium and acrylated-WBPUs

Introduction. Supraspinatus tears comprise most rotator cuff injuries, the leading cause of shoulder pain and an increasing problem with ageing populations. Surgical repair of considerable or persistent damages is customary, although not invariably successful. Tissue engineering presents a promising alternative to generate functional tissue constructs with improved healing capacities. This study explores tendon tissue constructs’ culture in a platform providing physiological mechanical stimulation and reports on the effect of different loading regimes on the viability of human tendon cells. Method. Porcine decellularized tendon scaffolds were fixed into flexible, self-contained bioreactor chambers, seeded with human tenocytes, allocated in triplicates to either static control, low (15±0.8Newtons [N]), medium (26±0.5N), or high (49±2.1N)-force-regime groups, connected to a perfusion system and cultured under standard conditions. A humanoid robotic arm provided 30-minute adduction/abduction stimulation to chambers daily over a week. A metabolic activity assay served to assess cell viability at four time points. Statistical significance = p<0.05. Result. One day after beginning mechanical stimulation, chambers in the medium and high-force regimes displayed a rise in metabolic activity by 3% and 5%, respectively. By the last experimental day, all mechanical stimulation regimes had induced an augment in cell viability (15%, 57% and 39% with low, medium, and high loads, respectively) matched against the static controls. Compared to all other conditions, the medium-force regime prompted an increased relative change in metabolic activity for every time point set against day one (p<0.05). Conclusion. Human tenocytes’ viability reflected by metabolic activity in a physiologically relevant bioreactor system is enhanced by loading forces around 25N when mechanically stimulating using adduction/abduction motions. Knowing the most favourable load regime to stimulate tenocyte growth has informed the ongoing exploration of the distinctive effect of different motions on tendon regeneration towards engineering tissue grafts. This work was supported by the Engineering and Physical Sciences Research Council EP/S003509/1

Introduction. Achieving an appropriate primary stability after implantation is a prerequisite for the long-term viability of a dental implant. Virtual testing of the bone-implant construct can be performed with finite element (FE) simulation to predict primary stability prior to implantation. In order to be translated to clinical practice, such FE modeling must be based on clinically available imaging methods. The aim of this study was to validate an FE model of dental implant primary stability using cone beam computed tomography (CBCT) with ex vivo mechanical testing. Method. Three cadaveric mandibles (male donors, 87-97 years old) were scanned by CBCT. Twenty-three bone samples were extracted from the bones and conventional dental implants (Ø4.0mm, 9.5mm length) were inserted in each. The implanted specimens were tested under quasi-static bending-compression load (cf. ISO 14801). Sample-specific homogenized FE (hFE) models were created from the CBCT images and meshed with hexahedral elements. A non-linear constitutive model with element-wise density-based material properties was used to simulate bone and the implant was considered rigid. The experimental loading conditions were replicated in the FE model and the ultimate force was evaluated. Result. The experimental ultimate force ranged between 67 N and 789 N. The simulated ultimate force correlated better with the experimental ultimate force (R. 2. =0.71) than the peri-implant bone density (R. 2. =0.30). Conclusion. The developed hFE model was demonstrated to provide stronger prediction of primary stability than peri-implant bone density. Therefore, hFE Simulations based on this clinically available low-radiation imaging modality, is a promising technology that could be used in future as a surgery planning tool to assist the clinician in evaluating the load-bearing capacity of an implantation site. Acknowledgements. Funding: EU's Horizon 2020 grant No: 953128 (I-SMarD). Dental implants: THOMMEN Medical AG

Introduction. Within articular cartilage, chondrocytes reside within the pericellular matrix (PCM), collectively constituting the microanatomical entity known as a chondron. The PCM functions as a pivotal protective shield and mediator of biomechanical and biochemical cues. In the context of Osteoarthritis (OA), enzymatic degradation of the PCM is facilitated by matrix metalloproteinases (MMPs). This study delves into the functional implications of PCM structural integrity decline on the biomechanical properties of chondrons and impact on Ca. 2+. signaling dynamics. Method. Chondrons isolated from human cartilage explants were incubated with activated MMP-2, -3, or -7. Structural degradation of the pericellular matrix (PCM) was assessed by immunolabelling (collagen type VI and perlecan, n=5). Biomechanical properties of chondrons (i.e. elastic modulus (EM)) were analyzed using atomic force microscopy (AFM). A fluorescent calcium indicator (Fluo-4-AM) was used to record and quantify the intracellular Ca. 2+. influx of chondrons subjected to single cell mechanical loading (500nN) with AFM (n=7). Result. Each of the three MMPs disrupted the structural integrity of the PCM, leading to attenuated fluorescence intensity for both perlecan and collagen VI. A significant decrease of EM was observed for all MMP groups (p<0.005) with the most notable decrease observed for MMP-2 and MMP-7 (p<0.001). In alignment with the AFM results, there was a significant alteration in Ca. 2+. influx observed for all MMP groups (p<0.05), in particular for MMP-2 and MMP-7 (p<0.001). Conclusion. Proteolysis of the PCM by MMP-2, -3, and -7 not only significantly alters the biomechanical properties of articular chondrons but also affects their mechanotransduction profile and response to mechanical loading, indicating a close interconnection between these processes. These findings underscore the influence of an intact pericellular matrix (PCM) in protecting cells from high stress profiles and carry implications for the transmission of mechanical signaling during OA onset and progression

INTRODUCTION. Intervertebral disc (IVD) degeneration is not completely understood because of the lack of relevant models. In vivo models are inappropriate because animals are quadrupeds. IVD is composed of the Nucleus Pulposus (NP) and the Annulus Fibrosus (AF), an elastic tissue that surrounds NP. AF consists of concentric lamellae made of collagen I and glycosaminoglycans with fibroblast-like cells located between layers. In this study, we aimed to develop a novel 3D in vitro model of Annulus Fibrosus to study its degeneration. For this purpose, we reproduced the microenvironment of AF cells using 3D printing. METHOD. An ink consisting of dense collagen (30 mg.mL. -1. ) and tyramine-functionalized hyaluronic acid (THA) at 7.5 mg.mL. -1. was first designed by modulating pH and [NaCl] in order to inhibit the formation of polyionic complexes between collagen and THA. Then, composite inks were printed in different gelling baths to form collagen hydrogels. Last, THA photocrosslinking using eosin and green light was performed to strengthen hydrogels. Selected 3D printed constructs were then cellularized with fibroblasts. RESULTS. The physicochemical study revealed that collagen/THA solutions (4:1 ratio) used at pH 5 with 200 mM NaCl were homogenous. In addition, collagen fibrils were observed in these solutions. The dense composite collagen/THA inks printed in a 2X PBS bath rapidly gelled and the photo-crosslinking increased the mechanical properties by 2 to reach 25 kPa (Young's modulus). Then, 3D printing parameters were optimized (85 kPa, extrusion, 4.5 mm/s speed and 80% fill-in percentage) to generate flat and anisotropic lamellae observed by polarized light microscopy. For the in vitro study, several anisotropic layers were printed and fibroblasts seeded between them. Cells adhered to layers, spread, proliferate and aligned along the axis of printed layers. CONCLUSION. Taken together, these results show it is possible to reproduce in vitro the main AF's biochemical and physical properties

Introduction. The non-union of long bones poses a substantial challenge to clinicians and patients alike. The Ilizarov fixation system and Limb Reconstruction System (LRS), renowned for their versatility in managing complex non-unions. The purpose of this retrospective study was to assess the outcomes of acute docking with the bone peg-in-bone technique for the management of non-unions of long bones. The study seeks to evaluate its effectiveness in achieving complete bony union, preserving limb length and alignment, correcting existing deformities, and preventing the onset of new ones. Method. A retrospective analysis of 42 patients was done with infected and non-infected non-unions of long bones who received treatment at a tertiary care hospital between April 2016 to April 2022. We utilized the Association for the Study and Application of Methods of the Ilizarov (ASAMI) scoring system to assess both bone and functional outcomes and measured mechanical lateral distal femoral angle (mLDFA) for the femur and the medial proximal tibial angle (MPTA) for the tibia. Result. In our retrospective study involving 42 patients, a total of 30 patients had post debridement gap of >2 cm and average gap of 4.54 cm (range 1 – 13 cm) and therefore underwent corticotomy and lengthening. The average external fixation time was 6.52 (range 4 – 11 months) and average external fixation index of 2.08 (range 0.4 – 4.5 months/cm). The ASAMI scoring system showed bone result of 38 excellent, 3 good and 1 fair. Functional result of 40 excellent and 2 good outcomes. The post op mLDFA and MPTA were in normal range except in 3 patients which not statistically significant. Conclusion. In conclusion, the use of acute docking provides several advantages such as promoting early fracture healing, increasing stability, shortening treatment time, reducing the number of surgical procedures and reduced number of complications

Introduction. Degenerative meniscal tears are the most common meniscal lesions, representing huge clinical and socio-economic burdens. Their role in knee osteoarthritis (OA) onset and progression is well established and demonstrated by several retrospective studies. Effective preventive measures and non-surgical treatments for degenerative meniscal lesions are still lacking, also because of the lack of specific and accurate animal models in which test them. Thus, we aim to develop and validate an accurate animal model of meniscus degeneration. Method. Three different surgical techniques to induce medial meniscus degenerative changes in ovine model were performed and compared. A total of 32 sheep (stifle joints) were subjected to either one of the following surgical procedures: a) direct arthroscopic mechanical meniscal injury; b) peripheral devascularization and denervation of medial meniscus; c) full thickness medial femoral condyle cartilage lesion. In all the 3 groups, the contralateral joint served as a control. Result. From a visual examination of the knee joint emerged a clear difference between control and operated groups, in the menisci but also in the cartilage, indicating the onset of OA-related cartilage degeneration. The meniscal and cartilaginous lesions were characterized by different severity and location in the different groups. For instance, a direct meniscal injury caused cartilaginous lesions especially in the medial part of the condyles, and the other approaches presented specific signature. Evaluation of scoring scales (e.g. ICRS score) allowed the quantification of the damage and the identification of differences among the four groups. Conclusion. We were effectively able to develop and validate a sheep model of meniscal degeneration which led to the onset of OA. This innovative model will allow to test in a pre-clinical relevant setting innovative approaches to prevent meniscal-related OA. Funding. Project PNRR-MAD-2022-12375978 funded by Italian Ministry of Health

Introduction. Ink engineering can advance 3D-printability for better therapeutics, with optimized proprieties. Herein, we describe a methodology for yielding 3D-printable nanocomposite inks (NC) using low-viscous matrices, via the interaction between the organic and inorganic phases by chemical coupling. Method. Natural photocurable matrices were synthesized: a protein – bovine serum albumin methacrylate (BSAMA), and a polysaccharide – hyaluronic acid methacrylate (HAMA). Bioglass nanoparticles (BGNP) were synthesized and functionalized via aminosilane chemistry. The functionalization of BSAMA, HAMA, and BGNP were quantified via NMR. To arise extrudable inks, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) chemistry was used to link innate carboxylic groups of BSAMA/HAMA and amine-functionalized BGNP. Different crosslinker and BGNP amounts were tested. Visible light photopolymerization is performed, using lithium phenyl-2,4,6-trimethylbenzoylphosphinate. The NC's rheological, mechanical, and biological behavior was evaluated before 3D extrusion printability. Result. All composite formulations effectively immobilized and homogeneously dispersed the BGNP, turning low-viscous materials (< 1 Pa) into shear-thinning formulations with tunable increased elastic/viscous moduli (50-500 Pa). More pronounced increments were found with increasing EDC/NHS and BGNP concentrations. The resulting inks produce robust and stable scaffolds successfully retrieved after post-print photocrosslinking (1-5 kPa). Bioactivity in simulated body fluid and in vitro assays using adipose-derive stem cells revealed a similar calcium/phosphate ratio to that of hydroxyapatite, and increased viability and metabolic activity. BSAMA and HAMA demonstrated distinct natures not only in printability but also in overall cellular performance and mechanical properties, making these ideal for interfacial tissue engineering. Conclusion. This strategy demonstrated being effective and reproducible to advance nanocomposites for 3D printing using different types of biomaterials. Further, we envision using both inks to produce hierarchical constructs via extrusion printing, better mimicking bone-to-cartilage interfaces. Acknowledgements. FCT grants (DOI:10.54499/2022.04605.CEECIND/CP1720/CT0021), (BI/UI89/10303/2022), (PRT/BD/154735/2023); EU's Horizon 2020 research and innovation programs InterLynk (Nº953169) and SUPRALIFE (Nº101079482) projects; CICECO-Aveiro Institute of Materials projects (DOI:10.54499/UIDB/50011/2020), (DOI:10.54499/UIDP/50011/2020), and (DOI:10.54499/LA/P/0006/2020), financed by FCT/MCTES(PIDDAC)

Introduction. Immunomodulation represents a novel strategy to improve bone healing in combination with low doses of bone morphogenetic growth factors like BMP-2. This study aims to investigate the effect and timing of monoclonal anti-IL-1ß antibody administration with 1μg BMP-2 on bone healing over 14 weeks in a rat femur segmental defect model. Method. 2 mm femoral defects were created in 22-27 weeks-old female Fischer F344 rats, internally fixed with a plate (animal license: GR/19/2022) using established protocols for analgesia and anesthesia. Animals (n=4/group) received either a collagen sponge, a collagen sponge+1μg BMP-2 (InductOs, Medtronic) or a collagen sponge+1μg BMP-2 with a monoclonal anti-IL-1ß antibody (BioXCell, 10 mg/ml), administered intravenously under anesthesia every third day until day 15, from day 0 or 3. In vivo micro-CT was performed after surgery and at 2, 3, 4, 6, 8, 10 and 14-weeks post-OP. Mechanical properties of the operated femurs were assessed by 4-point bending (Instron5866) and compared to contralateral femurs (one-way ANOVA, GraphPad Prism8). Histopathological analysis was performed semi-quantitatively on Giemsa-Eosin-stained sections (Olympus BX63) using a six-grade severity grading scale. Result. Operated femurs with BMP-2 reached an average stiffness of 91±37% of contralateral femurs, femurs in IL-1ß groups 105±11% (day 0) and 111±12% (day 3). Administration of anti-IL-1ß+1μg BMP-2 led to faster cortical bridging (3/4 femurs bridged by week 4 for day 0, 4/4 for day 3) than 1μg BMP-2 alone (0/4 by week 4). Micro-CT results confirmed histopathological evaluation, as collagen sponge alone led to non-union, complete bicortical bridging was observed for 3/4 femurs in the BMP-2 group and for 4/4 femurs in the IL-1β groups after 14 weeks. Conclusion. Anti-IL-1ß had a beneficial effect on late fracture healing with faster cortical bridging and new bone formation than 1μg BMP-2 alone. Acknowledgments. AO foundation. We thank Andrea Furter, Alisa Hangartner and Thomas Krüger for technical support

Introduction. The incidences of fragility fractures, often because of osteoporosis, are increasing. Research has moved towards bioresorbable scaffolds that provide temporary mechanical stability and promote osteogenesis. This research aims to fabricate a 3D printed composite Poly (l-lactic-co-glycolic acid)-strontium doped tricalcium phosphate (PLGA-SrTCP) scaffold and evaluate in an in vitro co culture study containing osteoporotic donor cells. Method. PLGA, PLGA TCP, and PLGA SrTCP scaffolds were produced using Fused Filament Fabrication (FFF). A four-group 35-day cell culture study was carried out using human bone marrow derived mesenchymal stem cells (hMSCs) from osteoporotic and control donors (monoculture) and hMSCs & human monocytes (hMCs) (Co culture). Outcome measures were biochemical assays, PCR, and cell imaging. Cells were cultured on scaffolds that had been pre-degraded for six weeks at 47°C prior to drying and gamma sterilisation. Result. 3D printed scaffolds were successfully produced by FFF. All groups in the study supported cell attachment onto the scaffolds, producing extracellular matrices as well as evidence of osteoclast cell structures. Osteoporotic cells increased CTSK activity and CAII activity and decreased ALP activity compared to controls. In control cultures, the addition of bTCP and bTCP/Sr to the PLGA reduced TRAP5b, CAII and ALP activity compared to PLGA alone. The addition of Sr did not show any differences between donors. Conclusion. This study details suitability of 3D printed polymer scaffolds for use in bone tissue applications. Both composite and pure polymer scaffolds promote osteogenesis in vitro. The introduction of ceramic filler and ion doping does not beneficially effect osteogenic potential and can reduce its ability compared to pure polymer. This study suggests the behaviour of control and osteoporotic cells are different and that osteoporotic cells are more prone to bone resorption. Therefore, it is important to design bone scaffolds that are specific to the patient as well as to the region of fracture

Introduction. Osteoarthritis (OA) often results from joint misloading, which affects chondrocyte calcium signaling through mechano-sensitive receptors such as Piezo1, -2, and TRPV4. Activation of Piezo1, especially under inflammatory conditions, can trigger premature chondrocyte apoptosis. Intra-articular glucocorticoid therapy, while beneficial against inflammation and pain in osteoarthritis, may induce oxidative stress and chondrotoxicity at higher doses. This study aims to assess the effects of glucocorticoids, particularly triamcinolone, on chondrocyte elasticity and mechanosignaling. Method. Chondrocytes isolated from articular condyles obtained from patients undergoing knee replacement surgery (n= 5) were cultured for 7 days in triamcinolone acetonide (TA) at different concentrations (0.2µM – 2mM). Cytoskeletal changes were assessed by F-actin labeling. Cell elasticity was measured using atomic force microscopy (AFM). Labeling cells (n=6 patients) with the calcium-sensitive dye (Fluo-4) enabled monitoring changes in intracellular calcium fluorescence intensity during guided single-cell mechanical indentation (500 nN) by AFM. Result. Cell exposure to 2 mM TA led to cell death and crystallization of TA in the cell culture media. However, the concentration of TA for intra-articular application is 46 times higher at 92.1 mM (40 mg/ml). The maximal pharmacological effect on viable cells was observed at 0.2 mM. AFM results showed a significant decrease of elasticity (p<0.001), alongside significantly higher calcium intensities both prior to and during mechanical stimulation in the TA-treated samples (p<0.05). Conclusion. Administration of TA significantly impacts the mechanical properties of chondrocytes, reducing cellular elasticity while simultaneously enhancing calcium-dependent mechanosensitivity. This data suggests a correlation between glucocorticoid-induced changes in cell elasticity and cell mechanosensitivity. Finding ways to minimize the effect of glucocorticoids on cell mechanosensitivity could help to make future therapies safer and reduce side effects

Introduction. Piezo1 is a mechanosensitive Ca. 2+. ion channel that has been shown to transduce hyper-physiologic mechanical loads in chondrocytes. In osteoarthritic cartilage, Piezo1 expression was shown to be upregulated by interleukin-1 alpha (IL-1α) and resulted in altered calcium dynamics and actin cytoskeleton rarefication. Together these studies highlight the importance of Piezo1 channels during joint injury. However, the mechanism by which Piezo1 regulates chondrocyte physiology and mechanotransduction during homeostasis is still largely unknown. In this study, we investigate the impact of Piezo1 activation on nuclear mechanics and chromatin methylation state. Methods. Porcine chondrocytes (n=3-5 pigs) were treated with Yoda1, a Piezo1-specific agonist, for either 2, 5, 15 or 180 minutes. To characterize chromatin state, we monitored the abundance of a chromatin methylation marker (H3K9Me3) using immunofluorescence (IF). Atomic force microscopy (AFM, 25 nm cantilever) was employed to quantify the nuclear elastic modulus (NEM) of individual cell nuclei. To explore the interplay between cytoskeletal dynamics and nuclear mechanics, chondrocytes were treated with Latrunculin A (LatA), an actin polymerization inhibitor. Result. IF experiments showed chromatin methylation was the lowest 2 minutes post Yoda1 activation of Piezo1 (p=0.027). Additionally, we found that 2 or 5 minutes post-Piezo1 activation resulted in a significantly lower NEM when compared to the control (p<0.00001). The observed decrease in NEM at 2 and 5 minutes post-Piezo1 activation was not observed after knocking down Piezo1 (p>0.99). In LatA treated cells, the elevated NEM persisted even after Piezo1 activation with Yoda1 (p>0.75). Conclusion. These findings illuminate the mechanism by which Piezo1 activation and actin remodeling regulate transient mechanotransduction during homeostasis. Further research into the transient decrease in nuclear stiffness and chromatin methylation observed during the initial 5 minutes of Piezo1-induced Ca2+ signaling, may contribute to a better understanding of the role of Piezo1 channels in joint injury and development of therapeutic interventions for osteoarthritis

Aims. For rare cases when a tumour infiltrates into the hip joint, extra-articular resection is required to obtain a safe margin. Endoprosthetic reconstruction following tumour resection can effectively ensure local control and improve postoperative function. However, maximizing bone preservation without compromising surgical margin remains a challenge for surgeons due to the complexity of the procedure. The purpose of the current study was to report clinical outcomes of patients who underwent extra-articular resection of the hip joint using a custom-made osteotomy guide and 3D-printed endoprosthesis. Methods. We reviewed 15 patients over a five-year period (January 2017 to December 2022) who had undergone extra-articular resection of the hip joint due to malignant tumour using a custom-made osteotomy guide and 3D-printed endoprosthesis. Each of the 15 patients had a single lesion, with six originating from the acetabulum side and nine from the proximal femur. All patients had their posterior column preserved according to the surgical plan. Results. Postoperative pathological assessment revealed a negative surgical margin was achieved in all patients. At final follow-up, 13.3% (2/15) died and no recurrence occurred. The overall survival was 81.7% at five years. None of the patients showed any signs of aseptic loosening, and no wound healing issues were observed. In total, 20% (3/15) developed complications, with two cases of early hip dislocation and one case of deep infection. The cumulative incidence of mechanical and non-mechanical failure in this series was 13.7% and 9.3%, respectively, at five years. In this cohort, the mean time to full weightbearing was 5.89 (SD 0.92) weeks and the mean Musculoskeletal Tumor Society score was 24.1 (SD 4.4). Conclusion. For patients with a hip joint tumour who met the inclusion criteria and were deemed suitable for posterior column preservation, a custom-made osteotomy guide combined with 3D-printed endoprosthesis is worth performing when treating patients who require extra-articular resection of the hip joint, as it can achieve adequate margin for local control, maximize bone preservation to maintain pelvic ring integrity, reduce the risk of complications by simplifying the surgical procedure, and allow for more precise reconstruction for better function. Cite this article: Bone Jt Open 2024;5(11):1027–1036

Aims

Distal femoral osteotomies (DFOs) are commonly used for the correction of valgus deformities and lateral compartment osteoarthritis. However, the impact of a DFO on subsequent total knee arthroplasty (TKA) function remains a subject of debate. Therefore, the purpose of this study was to determine the effect of a unilateral DFO on subsequent TKA function in patients with bilateral TKAs, using the contralateral knee as a self-matched control group.