Summary Statement. This study demonstrated that Sclerostin monoclonal antibody (Scl-Ab) enhanced bone healing in the rat osteotomy model. Scl-Ab increased callus size, callus bone volume fraction, rate of callus bone formation and fracture callus strength. Introduction. Sclerostin is a protein secreted by osteocytes and is characterized as a key inhibitor of osteoblast-mediated bone formation. Previous studies demonstrated that treatment with a sclerostin monoclonal antibody (Scl-Ab) results in significantly increased bone formation, bone mass and strength in rat closed fracture model (1–2). However, the effects of Scl-Ab on healing of open fracture model have not yet been reported in rats. Previously in ORS and ASBMR Annual Meeting, we have reported that Scl-Ab promoted the open fracture healing at week 3 and week 6 post-fracture. Here we extended our investigation for up to week 9 with additional histological assessments and dynamic histomorphometric analysis to investigate the effects of systemic administration of Scl-Ab on a later phase of fracture repair. Patients & Methods. Animal research ethics approval was obtained from our institute (reference No. 09/042/MIS), and the institute's guidelines for the care and use of laboratory animals were followed. In total, 120 six-month-old male SD rats were randomly divided into Scl-Ab group and vehicle group after a transverse osteotomy performed at the mid-shaft of right femur with internal fixation. One day post-surgery, rats were treated with a rodent Scl-Ab (Scl-Ab IV, s.c. injection, 25 mg/kg, 2 times per week) or vehicle for 3, 6 or 9 weeks. The progress of fracture healing for each animal was monitored weekly by digital radiography. Images acquired 3, 6 and 9 weeks post-operation were analyzed by ImageJ to quantify the total area of the fracture calluses. After euthanasia, femora were collected and subjected to the following analyses: micro-CT for bone mineral density (BMD) and callus volume fraction (BV/TV), micro-CT-based angiography for angiogenesis, histological evaluation and dynamic histomorphometry, and four-point mechanical testing for ultimate load, energy to failure and stiffness (3–6). Two-way ANOVA with Bonferroni post-hoc test was used to analyze the data. Significance level was set at P<0.05. Results. Radiographically, Scl-Ab treatment groups had significantly larger fracture calluses compared with respective vehicle group starting from week 3 post-fracture by quantitative analysis. Micro-CT analysis showed that Scl-Ab treatment groups had significantly higher callus bone volume fraction (+16–23%, P<0.01) and BMD (+15–16%, P<0.01) compared with respective vehicle groups at all time points post-fracture. Histological analysis also revealed more bone and less cartilage tissue in calluses in Scl-Ab group starting at week 3, which is explained by faster in the rate of new bone formation in fluorescence microscopy. Micro-CT based angiography demonstrated that Scl-Ab significantly enhanced neovasculation at the fracture calluses at week 3. Four-point bending test showed significantly higher ultimate load in Scl-Ab group than vehicle group at week 6 (+98%, P<0.01) and week 9 (+45%, P<0.05) post-fracture. In addition, ultimate load at week 6 of Scl-Ab group was at the similar level as seen at week 9 of the vehicle group, indicating the increased healing by Scl-Ab in this model. Stiffness (week 6 and 9) and energy to failure (week 6) were also tended higher in Scl-Ab group. Discussion/Conclusion. This study demonstrated that Scl-Ab enhanced bone healing in the rat osteotomy model. Scl-Ab increased callus size, callus bone volume fraction, rate of callus bone formation and fracture callus strength. Neovasculation was enhanced in the Scl-Ab group at week 3, implying Scl-Ab may enhance coupling of osteogenesis and angiogenesis. Scl-Ab treatment also resulted in more bone and less cartilage tissue in fracture calluses. Our results indicated that the systemic administration of Scl-Ab enhanced open fracture healing in rat femoral osteotomy model

Increased revision rates and early failure of Metal-on-Metal (MoM) hip replacements are often due to adverse reaction to metal debris (ARMD). Cobalt is a major component of MoM joints and can initiate an immune response via activation of the innate immune receptor Toll-like receptor 4 (TLR4). This leads to increased secretion of inflammatory cytokines/chemokines e.g. CCL3 and CCL4. The aim of this study was to evaluate whether TLR4-specific neutralising antibodies can prevent cobalt-mediated activation of TLR4. MonoMac 6 (MM6) cells, a human macrophage cell line, were treated with two different TLR4-specific monoclonal antibodies followed by 0.75mM of cobalt chloride (CoCl2). Lipopolysaccharide (LPS), a known TLR4 agonist was used as a positive control. Enzyme-linked immunosorbent assay (ELISA) was used to assess CCL3/CCL4 protein secretion and real time- polymerase chain reaction (RT-PCR) allowed quantification of CCL3/CCL4 gene expression. MM6 cells treated with cobalt and LPS up-regulate CCL3 and CCL4 gene expression and protein secretion. MM6 cells pre-treated with both monoclonal antibodies prior to stimulation with 0.75mM CoCl2 for 16 hours demonstrated significant inhibition of both CCL3 and CCL4 secretion as well as gene expression (both p=<0.0001). One of the antibodies failed to inhibit chemokine expression and secretion in LPS treated cells. This study identifies for the first time the use of TLR4-specific monoclonal antibodies to prevent cobalt activation of TLR4 and subsequent inflammatory response. This finding demonstrates the potential to exploit TLR4 inhibition in the context of MoM joint replacements by contributing to the development of novel therapeutics designed to reduce the incidence of ARMD

Aim. Implant infections caused by Staphylococcus aureus are difficult to treat due to biofilm formation, which complicates surgical and antibiotic treatment. Herewith we introduce an alternative approach using monoclonal antibodies (mAbs) targeting S. aureus and provide the biodistribution and specificity in a mouse implant infection model. Methods. 4497-IgG1targeting S. aureus Wall Teichoic Acid was labeled to Indium-111 using “CHXA” as a chelator. SPECT-CT scans were performed at 24, 72 and 120 hours after administration in Balb/cAnNCrl mice with a subcutaneous implant pre-colonized with biofilm of S. aureus. Biodistribution over the various organs of this labelled antibody was visualized and quantified using SPECT-CT imaging and compared to uptake at the target tissue with implant infection. Results. Uptake of the . 111. In-4497 mAbs (half-life 59 hours) at the infected implant gradually increased from 8.34%ID/g at 24 hours to 9.22%ID/g at 120 hours. Uptake at the heart/blood pool decreased over time from 11.60 to 7.58%ID/g whereas the uptake in other organs decreased from 7.26 to less than 4.66%ID/g at 120 hours. Conclusion. 111. In-4497 mAbs was found to specifically detect S. aureus and its biofilm with excellent and prolonged accumulation at the colonized implant site. Therefore, it holds great promise as a drug delivery system for diagnostic and bactericidal treatment of biofilm. However, high activity in the blood pool must be considered as it could pose a risk to healthy tissue

Objectives. The lack of effective treatment for cartilage defects has prompted investigations using tissue engineering techniques for their regeneration and repair. The success of tissue-engineered repair of cartilage may depend on the rapid and efficient adhesion of transplanted cells to a scaffold. Our aim in this study was to repair full-thickness defects in articular cartilage in the weight-bearing area of a porcine model, and to investigate whether the CD44 monoclonal antibody biotin-avidin (CBA) binding technique could provide satisfactory tissue-engineered cartilage. Methods. Cartilage defects were created in the load-bearing region of the lateral femoral condyle of mini-type pigs. The defects were repaired with traditional tissue-engineered cartilage, tissue-engineered cartilage constructed with the biotin-avidin (BA) technique, tissue-engineered cartilage constructed with the CBA technique and with autologous cartilage. The biomechanical properties, Western blot assay, histological findings and immunohistochemical staining were explored. Results. The CBA group showed similar results to the autologous group in biomechanical properties, Moran’s criteria, histological tests and Wakitani histological scoring. Conclusions. These results suggest that tissue-engineered cartilage constructed using the CBA technique could be used effectively to repair cartilage defects in the weight-bearing area of joints. Cite this article: H. Lin, J. Zhou, L. Cao, H. R. Wang, J. Dong, Z. R. Chen. Tissue-engineered cartilage constructed by a biotin-conjugated anti-CD44 avidin binding technique for the repairing of cartilage defects in the weight-bearing area of knee joints in pigs. Bone Joint Res 2017;6:–295. DOI: 10.1302/2046-3758.65.BJR-2016-0277

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: Monoclonal antibodies (mAbs) recognizing linear sulphation motifs in keratan sulphate (KS) were first developed in the early 1980’s. Over the years, ELISAs using 5-D-4 or other related anti-KS mAbs have been used in many studies monitoring increased cartilage aggrecan degradation with the onset of degenerative joint diseases. However, whilst these studies have in general been useful for monitoring some aspects of disease progression (usually in parallel with other biomarker assays), many longitudinal studies have shown efficacy in only the transient (early, mid or late) stages of the degenerative joint disease process. During the onset of degenerative joint disease, the pathological tissue attempts to repair/regenerate the cartilage, the chondrocytes thus synthesizing cartilage aggrecan with KS substitution [and chondroitin sulphate (CS) isomer composition] that is more like that found in developing or immature cartilage. This immature cartilage aggrecan contains much less KS substitution with shorter chain size and less linear sulphation motifs. Thus, during the different stages of degenerative joint disease progression one would expect to find variable changes in different linear sulphation epitopes present in the serum or synovial fluids. The aim of this study was to investigate the use of several monoclonal antibodies that recognise different sulphation epitopes [high sulphation (5-D-4), low sulphation (1-B-4) and KS-stubs (BKS-1)] to see if patterns of their expression could be used to distinguish different stages of degenerative joint disease. We have also developed ELISAs using mAbs recognising the KS-proteoglycans, keratocan (Ker 1) and lumican (Lum 1) for their quantification as potential biomarkers of osteoarthritis. Methods: Competitive ELISAs were developed using monoclonal antibodies (mAbs) 5-D-4, 1B4, BKS-1, Ker-1 and Lum-1. Bovine corneal KS-proteoglycans pre-treated with keratanase were used as both the coating antigen and “standard” antigen on the same ELISA plate. Blood, synovial fluid and cartilage samples (surgical waste) obtained from patients undergoing arthroplasty with different Kellgren & Lawrence grades were analysed. Results and Discussion: 5-D-4 and BKS-1 showed similar inhibition curves and relative 50% inhibition points. However, the curve obtained with 1B4 indicated lower relative expression of 1B4 epitope. Analysis of serum and synovial fluid sample with 5-D-4 mAb showed the presence of the epitope in both samples, but there was significantly less KS in serum than in the synovial fluid. Our results show that competitive ELISA for quantification of several different KS sulphation or “stub” epitopes and two KS-proteoglycans can all be quantified and compared using the same experimental conditions. These studies are ongoing as part of an Arthritis Research Campaign (UK) funded study. In addition the data indicates that keratocan and lumican are also increased in their expression with the progression of disease. Future studies will be performed in an attempt to quantify increased keratocan and lumican expression as potential biomarkers of degenerative joint disease

Deriving autologous mesenchymal stem cells (MSCs) from adipose tissues without using enzymes requires sophisticated biomedical instruments. Applied pressure on tissues and cells are adjusted manually although centrifugation and filtration systems are frequently used. The number of derived MSCs therefore could differ between instruments. We compared the number of MSCs obtained from four commercially available devices and our newly designed and produced instrument (A2, B3, L3, M2 and T3). Three-hundred mL of adipose tissue was obtained from a female patient undergoing liposuction using the transillumination solution. Obtained tissue was equally distributed to each device and handled according to the producers' guides. After handling, 3 mL stromal vascular fraction (SVF) was obtained from each device. Freshly isolated SVF was characterized using multi-color flow cytometry (Navios Flow Cytometer, Beckman Coulter, USA). Cell surface antigens were chosen according to IFATS and ISCT. CD31-FITC, CD34-PC5,5, CD73-PE, CD90-PB and CD45-A750 (Backman Coulter, USA) fluorochrome-labeled monoclonal antibodies were assessed. Markers were combined with ViaKrome (Beckman Coulter, USA) to determine cell viability. At least 10. 5. cells were acquired from each sample. A software (Navios EX, Beckman Coulter, USA) was used to create dot plots and to calculate the cell composition percentages. The data was analyzed in the Kaluza 2.1 software package (Beckman Coulter, USA). Graphs were prepared in GraphPad Prism. CD105 PC7/CD31 FITC cell percentages were 23,9%, 13,5%, 24,6%, 11,4% and 28,8% for the A2, B3, L3, M2 and T3 devices, respectively. We conclude that the isolated MSC percentage ranged from 11,4% to 28,8% between devices. The number of MSCs in SVF are key determinants of success in orthobiological treatments. Developing a device should focus on increasing the number of MSCs in the SVF while preserving its metabolic activity. Acknowledgments: Scientific and Technological Research Council of Türkiye (TÜBİTAK)- Technology and Innovation Funding Program Directorate (TEYDEB) funded this project (#321893). Servet Kürümoğlu and Bariscan Önder of Disposet Ltd., Ankara, Türkiye (. www.disposet.com. ) contributed to the industrial design and research studies. Ali Tuncel and Feza Korkusuz are members of the Turkish Academy of Sciences (TÜBA). Nilsu Baysal was funded by the STAR Program of TÜBITAK Grant # 3210893

Tendon diseases are prevalent health concerns for which current therapies present limited success, in part due to the intrinsically low regenerative ability of tendons. Therefore, tissue engineering presents a potential to improve this outcome. Here, we hypothesize that a concurrent control over both biophysical and biochemical stimuli will boost the tenogenic commitment of stem cells, thus promoting regeneration. To achieve this, we combine molecularly imprinted nanoparticles (MINPs), which act as artificial amplifiers for endogenous growth factor (GF) activity, with bioinspired anisotropic hydrogels. 2. to manufacture 3D tenogenic constructs. MINPs were solid phase-imprinted using a TGF-β3 epitope as template and their affinity for the target was assessed by SPR and dot blot. Magnetically-responsive microfibers were produced by cryosectioning electrospun meshes containing iron oxide nanoparticles. The constructs were prepared by encapsulating adipose tissue-derived stem cells (ASCs), microfibers, and MINPs within gelatin hydrogels, while aligning the microfibers with an external magnetostatic field during gelation. This allows an effective modulation of hydrogel fibrillar topography, mimicking the native tissue's anisotropic architecture. Cell responses were analyzed by multiplex immunoassay, quantitative polymerase chain reaction, and immunocytochemistry. MINPs showed an affinity for the template comparable to monoclonal antibodies. Encapsulated ASCs acquired an elongated shape and predominant orientation along the alignment direction. Cellular studies revealed that combining MINPs with aligned microfibers increased TGF-β signaling via non-canonical Akt/ERK pathways and upregulated tendon-associated gene expression, contrasting with randomly oriented gels. Immunostaining of tendon-related proteins presented analogous outcomes, corroborating our hypothesis. Our results thus demonstrate that microstructural cues and biological signals synergistically direct stem cell fate commitment, suggesting that this strategy holds potential for improving tendon healing and might be adaptable for other biological tissues. The proposed concept highlights the GF-sequestering ability of MINPs which allows a cost-effective alternative to recombinant GF supplementation, potentially decreasing the translational costs of tissue engineering strategies. Acknowledgements: The authors acknowledge the funding from the European Union's Horizon 2020 under grant No. 772817; from FCT/MCTES for scholarships PD/BD/143039/2018 & COVID/BD/153025/2022 (S.P.B.T.), and PD/BD/129403/2017 (S.M.B.), co-financed by POCH and NORTE 2020, under the Portugal 2020 partnership agreement through the European Social Fund, for contract 2020.03410.CEECIND (R.M.A.D.) and project 2022.05526.PTDC; and from Xunta de Galicia for grant ED481B2019/025 (A.P.)

Aim. Prosthetic joint infections (PJI) remain a great challenge in orthopedic surgery with a high mortality rate. It is particularly complicated by biofilms and infections caused by Methicillin-resistant Staphylococcus aureus (MRSA). It concurrently shields bacteria from host immune responses and confers resistance to antibiotics. This study aims to investigate the efficacy of radioimmunotherapy as an innovative therapeutic modality to address the challenges posed by MRSA and its biofilm. Method. We induced specific monoclonal antibodies 4497-IgG1 as carriers, which target wall teichoic acids (WTA) existing on MRSA and its biofilm. Radionuclides actiniumr-225 (. 225. Ac, α-emitter) and lutetium-177 (. 177. Lu, β-emitter) were conjugated with mAbs using DOTA as chelator. Quality control was assessed using thin layer chromatography and immunoreactivity assays. . 225. Ac- and . 177. Lu-labelled 4497-IgG1 were employed to evaluate the susceptibility of MRSA and its biofilm to the radioimmunotherapy in vitro. Planktonic MRSA and biofilms, at concentrations of 10. 8. and 10. 7. CFU/mL, were incubated at 37°C for 60 minutes in PBS containing either . 225. Ac-mAb (0 - 14.8 kBq) or . 177. Lu-mAb (0 - 14.8 MBq). Radiolabelled dunituximab and free radionuclides serve as isotype-matched negative control. The bacterial viability and metabolic activity were subsequently quantified using CFU and XTT assays. Results. The radiochemical purity of the . 225. Ac-mAbs and . 177. Lu-mAbs complex were determined to be 95.4% and 96.16%. Immunoreactivity fractions of them were measured at 81.8% and 80.8%. . 225. Ac-mAbs and . 177. Lu-mAbs exhibited significant and dose-dependent antimicrobial effects on both planktonic MRSA and biofilm. . 225. Ac- and . 177. Lu-4497IgG1 at doses of 7.4 kBq and 7.4 MBq resulted in more than 4-log reduction in bacterial counts. In biofilms, 2-log reduction at the highest . 225. Ac radioactivity of 14,8kBq. The . 177. Lu complex showed a strong dose-dependent effect, with a reduction of up to 4-log. The XTT assay confirmed these findings, showing a decrease in metabolic activity corresponding to a decrease in bacterial counts, and a slight increase in metabolic activity at the lower dose. Conclusions. Our study demonstrates the efficacy of . 225. Ac and . 177. Lu-labelled 4497-IgG1 antibodies in mediating dose-dependent bactericidal effects against planktonic MRSA and biofilms in vitro. This indicates that radioimmunotherapy could be a potential targeted therapeutic strategy against MRSA and its biofilm. Further research in preclinical and clinical settings is warranted to validate and refine these findings on biofilm-associated implant infections

Osteoarthritis (OA) is a major global disease with increasing prevalence. It is one of the most significant causes of disability worldwide and represents a major burden in terms of healthcare delivery and impact on the quality of life of patients. It is a cause of severe chronic pain and has given rise to alarming levels of opioid use and addiction. Despite this prevalence, there are no disease-modifying treatments which delay or reverse the degrative changes within joints which are characteristics of the disease. All treatments are symptom-modifying with the exception of joint arthroplasty, which is currently the most common surgical procedure carried out in US hospitals. Several pharmaceutical and biological interventions have been tested in recent years, including metalloproteinase inhibitors, chondrogenic agents such as Kartogenin, IL-1 antagonists and monoclonal antibodies. So far, none of these has provided an effective disease-modifying treatment. Cellular therapies have a great deal of promise because of their anti-inflammatory and regenerative effects. Mesenchymal stromal cells (MSCs) have been widely studied as a treatment for OA in preclinical and clinical assessments with generally positive results. As the clinical testing of these cells proceeds serious questions emerge relating to the quality and consistency of the therapeutic product and the need for better standardisation with regard to, for example, the tissue source and expansion conditions. Of equal importance is the need for deeper insight into the therapeutic mechanism, specifically the activity and phenotype of cells transplanted to the OA environment, their fate and interaction with local cells

To date there is no medical treatment alternative to surgery for osteolysis after THA. In this proof-of-concept clinical trial we examined the effect of a human monoclonal antibody against osteoclasts versus placebo on osteolytic lesion activity in patients undergoing revision surgery. Patients scheduled for revision for symptomatic osteolysis were randomised (1:1) to receive either denosumab 60mg or placebo subcutaneously eight weeks prior to operation. At surgery, biopsies from the osteolytic membrane-bone interface were taken for histomorphometric analysis of osteoclast number. Secondary outcome measures included systemic bone turnover markers. 22 subjects completed the study (10 denosumab). The denosumab group had 83% (−63 to −97), P=0.011 fewer osteoclasts at osteolytic lesion sites, 87% lower osteoclast surface (−65 to −95, P=0.009), and 72% lower eroded surface (−35 to −93, P=0.020) versus the placebo group. At surgery, serum CTX-I, TRAP5b and PINP were 80% (−65 to −95, p<0.001), 57% (−40 to −90, p<0.001), and 44% (−41 to −65, p<0.001) lower in the denosumab versus placebo groups, respectively. The rate of adverse events (denosumab 6, placebo 7) were similar between groups (P>0.05). These data provide a biological basis for a definitive clinical trial using pain, function and prosthesis survival as the study endpoints. As osteolysis/ aseptic loosening is the leading cause of prosthesis failure world-wide, the establishment of a non-surgical solution would reduce patient suffering and dramatically reducing the cost to healthcare economies

In a recent phase 2 superiority clinical trial we demonstrated that a single dose of 60mg of the human monoclonal antibody denosumab inhibits osteolytic lesion activity in patients undergoing revision total hip arthroplasty (THA), demonstrating proof of biological efficacy for this clinical application. Here, we examined the effect that denosumab has on disease biology at the osteolysis tissue level. Osteolytic tissue taken from the prosthesis-bone lesion interface at revision surgery in patients with osteolysis (n=10 participants that had received a single 60 mg dose of denosumab 8 weeks prior to revision surgery and n=10 that had received placebo) was examined for total genetic message activity and protein levels using whole genome sequencing and mass spectrometry, respectively. The top five upregulated enriched pathways with denosumab treatment included inflammatory response, myeloid cell activation, myeloid leukocyte migration, neutrophil and granulocyte activation (p<6.26 × 10. −28. ). Cell morphogenesis was amongst the most downregulated pathways (p<3.42 ×10. −23. ). Finally, comparison of the trial mRNA and protein data versus mouse single cell RNA sequencing data of the same pathway blockade in mouse tibia showed the same direction of effect, suggesting that giving the drug causes then cells responsible for osteolysis to disperse into a more immature form (128 of 189 genes (z=4.87, P<0.0001) disease and functional pathways at the mRNA level and 10 of 11 (z=2.72, P=0.0065) at the protein level). In this first-in-man study we identify multiple genes and pathways within periprosthetic osteolysis tissue that are affected by denosumab treatment. The dominant pathways involved upregulation of innate inflammatory signaling and downregulation of cell morphogenesis. We also found enrichment of similar disease and functional pathways at both the mRNA and protein levels versus mRNA pathway enrichment found in mouse osteomorphs. These data provide the first human data of the mechanistic effect of denosumab treatment on inflammatory osteolytic lesion activity after joint replacement that is necessary to support its clinical application. ∗Winner of The Bone & Joint Journal prize∗

Fragmentation of SLRPs, including decorin, biglycan, lumican, keratocan and fibromodulin, has been shown to occur in osteoarthritic articular cartilage. We have previously shown an increased expression of lumican and keratocan, in osteoarthritic articular cartilage. The long-term aim of this project is to develop ELISAs for the detection of SLRP metabolites, and validate these potential biomarkers with synovial fluid and serum samples from a large cohort of normal and osteoarthritic patients. Initially, we aimed to determine whether SLRPs could be detected in synovial fluid and whether they were post-translationally modified with glycosaminoglycan (GAG) attachments; and whether bovine nasal cartilage (BNC) would be a plentiful source of native SLRP for ELISA development. Proteoglycans were extracted from BNC in guanidine hydrochloride. BNC extract and bovine synovial fluid was separated on an associative CsCl gradient. BNC CsCl cuts containing sulphated GAG were further purified using anion exchange chromatography. SLRPs in each fraction were detected using Western Blotting. Human recombinant lumican was expressed in Chinese hamster ovary (CHO) cells. Monoclonal antibodies that recognise epitopes on the core protein of human and bovine lumican and decorin were purified from hybridoma media using Protein G and Protein A affinity chromatography respectively. Monoclonal antibody activity against native and recombinant SLRPs was then determined using a direct ELISA. Preliminary tests showed that bovine synovial fluid contains keratocan and lumican with GAG attachments. BNC is a good source of post-translationally modified decorin, keratocan and biglycan but lumican was present predominantly without GAG attachments. Human recombinant lumican was successfully expressed with GAG attachments by CHO cells. Initial tests showed that the mAb against decorin was able to detect native decorin, with GAG attachments, in direct ELISA conditions. We have identified a plentiful source of native SLRP and begun ELISA development to ascertain whether these proteoglycans are potential biomarkers of OA

Antigranulocyte scintigraphy with the use of monoclonal antibodies or antibody fragments (leukoscan) is a promising diagnostic tool that has been used widely during the recent years for the establishment of prosthesis infection after total hip or knee arthroplasty. In the present study we report the Ioannina University Hospital experience in the diagnostic efficacy of leukoscan for the diagnosis of prosthesis infection in patients with total hip or knee arthroplasty. We considered all patients with suspected total hip or knee arthroplasty infection that had undergone a leukoscan examination for the diagnosis of this infection in our department during the year 2004. All patients underwent a 3-phase bone scan and a subsequent leukoscan. For the whole group of patients the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy were calculated by comparison of the results of three-phase bone scan or leukoscan with the results obtained from cultures of synovial fluid obtained from articular puncture or cultures of intraoperative sampling from those patients that had undergone a revision surgery. A total of 19 patients with total joint arthroplasty (12 THA and 7 TKA) and suspected prosthesis infection were included in the study. The mean age of patients was 67 years. Twelve patients had verification of prosthesis infection in cultures. The overall sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy for the three-phase bone scan were 54%, 83%, 88%, 45% and 63% respectively. On the contrary, the overall sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy for leukoscan were 75%, 86%, 90%, 66% and 79%, respectively. Antigranulocyte scintigraphy with the use of monoclonal antibody fragment sulesomab (leukoscan) had good but not perfect diagnostic performance for the diagnosis of prosthesis infection in patients with total hip or knee arthroplasty

A fifty year old lady with history of rheumatoid arthritis (RA) for 24 years and COPD for 10 years was admitted for investigation of persistent chest infection and for the control of RA flare-up. She was on Sulphasalazine, NSAIDs and had completed a course of gold injections and on admission started on methotrexate, folic acid, Calcium, bisphosphonates and alendronate. Urinanalysis was positive for Bence Jones’ Proteins (BJP). Four days after admission patient developed spontaneous pain in the right thigh with inability to move the right leg. Radiographs showed a supracondylar femoral fracture through a lytic lesion, which was stabilised with a Distal femoral nail. At surgery bone quality of right femur was found to be very poor. Radiographs of the left femur showed a lytic lesion in the subtrochanteric region, which was stabilised prophylactically with a Proximal Femoral Nail. Histopathological examination of the marrow reamings from right femur showed no neoplastic changes and from left femur showed occasional plasma cells. 24 hour urinanalysis showed BJP of 0.22g/hour and protein electrophoresis showed monoclonal antibodies. Bone marrow biopsy was performed which showed only reactive cells. A week later 24 hour urine BJP was down to 0.13g/hour. At three weeks, symptoms of RA were under control and the protein electrophoresis showed no monoclonal banding. Chest infection resolved with appropriate antibiotics. Computerised Tomography of chest showed bronchiectasis with no evidence of neoplasm. Discussion: In acute stages of RA there is an increase in antibodies production that may present as positive for monoclonal antibodies on electrophoresis and by the same reason urine may be positive for BJP. The spontaneous fracture in this case could be due to severe reactive osteoporosis confounding the clinical picture of active RA. Without clear evidence of myeloma or plasmacytoma, instituting chemotherapy may lead to further complications in patients with RA

Sclerostin (SOST) is an endogenous inhibitor of Wnt/β-catenin signalling pathway to impair osteogenic differentiation and bone anabolism. SOST immunotherapy like monoclonal antibody has been observed to control bone remodeling and regeneration. This study is aimed to develop a SOST vaccine and test its protective effects on estrogen deficiency-induced bone loss in mice. Gene sequences coded SOST peptide putative targeting Wnt co-receptor LRP5 were cloned and constructed into vectors expressing Fc fragment to produced SOST-Fc fusion protein. Mice were subcutaneously injected SOST-Fc to boost anti-SOST antibody. Bone mineral density, microstructure, and mechanical property were quantified using μCT scanning and material testing system. Serum bone formation and resorption markers and anti-SOST levels were measured using ELISA. SOST-Fc injections significantly increased serum anti-SOST antibody levels but reduced serum SOST concentrations. SOST-Fc vaccination significantly reduced estrogen deficiency-induced serum bone resorption markers CTX-1 increased serum bone formation marker osteocalcin. Of note, it significantly alleviated the severity of estrogen-induced loss of bone mineral density, trabecular morphometric properties, and biomechanical forces of bone tissue. Mechanistically, SOSF-Fc vaccination attenuated trabecular loss histopathology and restored immunostaining of Wnt pathway like Wnt3a, β-catenin, and TCF4 in bone tissue along with increased serum osteoclast inhibitor OPG levels but decreased serum osteoclast enhancer RANKL concentrations. Taken together, SOST-Fc vaccination boosts anti-SOST antibody to neutralize SOST and mitigates the estrogen deficiency-induced bone mass and microstructure deterioration through preserving Wnt signalling. This study highlights an innovative remedial potential of SOST vaccine for preventing osteoporosis

Aim. “Implant associated Staphylococcus aureus or S. epidermidis infections are often difficult to treat due to the formation of biofilms on prosthetic material. Biofilms are bacterial communities adhered to a surface with a self-made extracellular polymeric substance that surrounds resident bacteria. In contrast to planktonic bacteria, bacteria in a biofilm are in an adherent, dormant state and are insensitive to most antibiotics. In addition, bacteria in a biofilm are protected from phagocytic cells of the immune system. Therefore, complete surgical removal and replacement of the prosthetic implant is often necessary to treat this type of infections. Neutrophils play a crucial role in clearing bacterial pathogens. They recognize planktonic bacteria via immunoglobulin (Ig) and complement opsonisation. In this project, we aim to evaluate the role of IgG and complement in the recognition and clearance of staphylococcal biofilms by human neutrophils. Furthermore, we evaluate if monoclonal antibodies (mAbs) targeting biofilm structures can enhance recognition and clearance of staphylococcal biofilms by the human immune system.”. Method. “We produced a set of 20 recombinant mAbs specific for staphylococcal antigens. Using flow cytometry and ELISA-based methods we determined the binding of these mAbs to planktonic staphylococci and in vitro staphylococcal biofilms. Following incubation with IgG/IgM depleted human serum we determined whether mAbs can react with the human complement system after binding to biofilm. Confocal microscopy was used to visualize the location of antibody binding in the biofilm 3D structure.”. Results. “We show that mAbs directed against several staphylococcal surface targets such as wall teichoic acid (a glycopolymer on the S. aureus/S. epidermidis cell wall) and polymeric-N-acetyl-glucosamine (major constituent of the S. epidermidis biofilm extracellular matrix) bind biofilms in a dose-dependent manner. This interaction was specific since no binding was observed for control antibodies (recognizing the hapten DNP). Furthermore we show that these antibodies can penetrate the complete 3D structure of an in vitro biofilm. Products of complement activation via the classical pathway were detected upon incubation with human serum and the biofilm binding mAbs.”. Conclusions. “Having established that our mAbs can bind biofilms and induce complement opsonisation via C3b deposition, we will now study if we can engineer these antibodies to enhance complement deposition. A combination of enhanced complement and antibody opsonisation may improve recognition and clearance of biofilms by phagocytic immune cells. These mAbs could be used to boost the immune system to clear implant associated infections, without the need to replace the implant via invasive surgical procedures.”

Background: The c-ebB-2 gene and its products (also designated HER-2 and c-neu) encode for a 185-kd transmembrane glycoprotein with intracellular tyrosine kinase activity. C-erbB-2 belongs to the epidermal growth factor receptor family, of which there are four known members, and has molecular homology to the epidermal growth factor receptor. It seems that this family is critical in control of growth, differentiation, and mobility of many normal and transformed epithelial cell types. Materials and Methods: We have looked for over expression of c-erbB-2 gene product in 230 cases of soft tissue sarcoma, in order to establish a possible new prognostic marker, and a potentially new treatment option. Results: In all the cases, irrespective of the sarcoma histological type, the immunostaining for erbB-2 was negative. Conclusions: Applications of erbB-2 for prognostication as well as the option of receptor targeting by trastuzumab monoclonal antibodies were aborted

The aim of this study was to find evidence of tissue hypoxia and apoptosis (programmed cell death) have on a human model of rotator cuff failure. We studied twenty seven patients with no tear mild impingment (3), no tear moderate impingment (3), no tear severe impingment (3), partial tear (3), small tear (3), moderate tear (3), large tear (3), massive tear (3) and control (3) who were undergoing shoulder arthroscopy, subacromial decompression and potential rotator cuff repair. A supraspinatus tendon biopsy was taken during debridement/repair on all cases (ethics number C01.071). Control tendon was obtained from the subscapularis tendon of patients undergoing stabilization surgery. Biopsies were analysed using two immunocytological techniques. A monoclonal antibody against BNIP-3 (a pro-apoptotic marker of hypoxia) and TUNEL (an apoptotic marker). An immunofluorescent nuclear counterstain DAPI (4 6-Diamidino-2-phenylindole dihy-drochloride) was used to stain all cells. Positive cells and total cell number were then counted in 10 high powered fields per section. The results showed a significant increase in BNIP-3 expression in the cuff tears compared with intact tendons. This increase was least in the massive tears. Apoptosis increases from mild impingement to massive cuff tears (mean 7.3% to 21%)

The aim of this study was to understand the role tissue hypoxia and apoptosis have on a human model of rotator cuff failure. We studied twenty seven patients with no tear mild impingment (3), no tear moderate impingment (3), no tear severe impingment (3), partial tear (3), small tear (3), moderate tear (3), large tear (3), massive tear (3) and control (3). A supraspinatus tendon biopsy was taken during debridement/repair in all cases (ethics no. C01.071). Control tendon was obtained from the subscapularis tendon of patients undergoing stabilization surgery. Biopsies were analysed using two immunocytological techniques. A monoclonal antibody against BNIP-III (a marker of hypoxia) and TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling – an apoptotic detection process). An immunoflorescent counterstain DAPI (4′,6-diamidino-2-phenylindol) was used to stain all cells. Positive cells and total cell number were then counted in 10 high powered fields. The results showed a significant increase in BNIP-III expression in the cuff tears compared with intact tendons. This increase was least in the massive tears. Apoptosis increases from mild impingment to massive cuff tears (mean 7.3% to 21%) In conclusion, as tear size increases, the viability of the tendon reduces with increasing hypoxia and apoptosis