Aims. Microbiological culture is a key element in the diagnosis of periprosthetic joint infection (PJI). However, cultures of periprosthetic tissue do not have optimal sensitivity. One of the main reasons for this is that microorganisms are not released from the tissues, either due to biofilm formation or intracellular persistence. This study aimed to optimize tissue pretreatment methods in order to improve detection of microorganisms. Methods. From December 2017 to September 2019, patients undergoing revision arthroplasty in a single centre due to PJI and aseptic failure (AF) were included, with demographic data and laboratory test results recorded prospectively. Periprosthetic tissue samples were collected intraoperatively and assigned to tissue-mechanical homogenization (T-MH), tissue-manual milling (T-MM), tissue-dithiothreitol (T-DTT) treatment, tissue-sonication (T-S), and tissue-direct culture (T-D). The yield of the microbial cultures was then analyzed. Results. A total of 46 patients were enrolled, including 28 patients in the PJI group and 18 patients in the AF group. In the PJI group, 23 cases had positive culture results via T-MH, 22 cases via T-DTT, 20 cases via T-S, 15 cases via T-MM, and 13 cases via T-D. Three cases under ongoing antibiotic treatment remained culture-negative. Five tissue samples provided the optimal yield. Any ongoing antibiotic treatment had a relevant influence on culture sensitivity, except for T-DTT. Conclusion. T-MH had the highest sensitivity. Combining T-MH with T-DTT, which requires no special equipment, may effectively improve bacterial detection in PJI. A total of five periprosthetic tissue biopsies should be sampled in revision arthroplasty for optimal detection of PJI. Cite this article: Bone Joint Res 2021;10(2):96–104

Aim. To evaluate clinical outcomes for patients with osteomyelitis at a major trauma centre limb reconstruction unit. Method. We prospectively evaluated 137 patients on the limb reconstruction database with long bone osteomyelitis. Data on initial diagnosis, management (bone resection, use of external fixation, dead space and soft tissue management), microbiology and 2-year outcomes were collated. 11 patients' data was incomplete and 9 underwent primary amputations; these were excluded from microbiology data analysis. The patient data was categorised into microbiological culture negative or culture positive groups. Inter-group comparisons were made to evaluate two-year outcomes and percentage failure rate. Results. Forty percent (55/137) of patients presented with infected non-union, 20% (27/137) infected fractures, 19% (26/137) chronic osteomyelitis without implants and 14% (19/137) had infected metalwork. Removal of metalwork, reaming and debridement were the most frequently performed procedures, often in combination. 3% of patients failed treatment and had persistent infected non-union. The most common microorganisms identified in the culture positive group were Staphylococcus aureus (47.6%), Coagulase Negative Staphylococcus species (11.9%) and Enterobacter cloacae (11.9%), however multiple organism growth was more common than single organism growth, 53% and 47% respectively. 8% of culture negative patients had histological evidence of infection on biopsy. Conclusions. The 2-year failure rate (persistent infective non-union) was higher in the culture negative group (8%) than the culture positive group (1%). The higher failure rate may be secondary to lack of organisms isolated and available sensitivities from deep tissue samples. In 9 cases patient preference led to primary amputation over limb salvage procedures, without further infection. Our work highlights the array of factors contributing to outcome in this patient group. The incidence of micro-organisms commonly encountered in this cohort will provide further evidence to support choice of antibiotic for empirical therapy especially in cases which are culture negative. Finally, there are many challenges in achieving adequate outcomes in patients with long bone infections thus the need for a multidisciplinary team approach in this patient cohort is invaluable. Routine histology testing may be beneficial as this may highlight infective processes in culture negative patents thereby allowing optimization of patient management

Aims. The microbiological detection of microorganisms plays a crucial role in the diagnosis as well as in the targeted systemic and local antibiotic therapy of periprosthetic infections (PJI). Despite extensive efforts to improve the sensitivity of current culture methods, the rate of culture-negative infections is approximately 10–20% of all PJI. This study investigates an preanalytical algorithm (culture collection and direct processing in the OR) to potentially increasing culture yield in patients with PJI. Methods. Patients undergoing staged revision arthroplasty for PJI in our hospital between October 2021 and 2022 were included in this prospective pilot study. Intraoperatively twenty tissue samples were collected and distributed among 4 groups. Tissue samples were prepared according to standard without medium and in thioglycolate medium at 3 different temperatures (room temperature, 4°C, 37° for 24h before transport to microbiology) directly in the OR. The removed implants were sonicated. Cultures were investigated on days 1, 3, 7, 12, 14 for possible growth. All grown organism, the number of positive samples and the time to positivity were recorded and compared. Results. 71 patients were included (age, gender). Compared to the standard procedure the thioglycolate broth at 37°C was significantly more often culture-negative (p=0.031). No significant differences in the frequency of culture-negative samples were detected in the other groups. 8.4% (6/71) patients were culture negative in the standard culture but positive in the thioglycolate samples. In contrast, 7% (5/71) were culture negative in the thioglycolate samples but had bacterial detection in the standard approach. In 4.7% (3/63) of the patients, only the sonication showed growth, whereas 25.4% (16/63) had no growth in sonication fluid but in one of the cultures. For S. caprae, there was a significantly different distribution (p=0.026) with more frequent detection in the group with thioglycolate at 37°C. The standard procedure (p=0.005) and sonication (p=0.023) showed a shorter time to positivity of the culture compared to the thioglycolate approach at 4°C. Conclusions. No general differences could be shown between the standard preparation and the thioglycolate preparation; in particular, storage at different temperatures does not seem to result in any difference. For individual cases (8% in this study), bacterial growth was detected in the thioglycolate group that would have been culture-negative otherwise. There might be organism dependent differences in growth in different media

Aim. Haematogenous prosthetic joint infections account for 20-35% of total prosthetic infections. Debridement, antibiotics and implant retention (DAIR) is a well-accepted treatment for these infections and probably the most desired by surgeons, since it tries to maintain a functional and stable implant. However, the risk of DAIR failure is not negligible and some risk factors have been described, and also, different scores, such as CRIME80. Nonetheless, less is known about the impact of positive blood cultures may have on DAIR treatment. The aim of our study is to analyze whether the presence of a positive culture is a risk factor for DAIR failure. Method. A retrospective cohort study of 50 late acute haematogenous TKA infections was performed from 2015 to 2023. DAIR failure was defined as the need of a subsequent intervention either a new DAIR or a revision surgery. So, patients were divided into two groups depending on the surgical outcome: successful (SG) vs failure (FG). Demographic variables including age, gender, affected side and body mass index were collected. Patient's comorbidities were also collected including chronic obstructive pulmonary disease (COPD), diabetes, rheumatoid arthritis (RA), cirrhosis and chronic renal failure, etc. Other variables, such as ones included in CRIME80 (C-reactive protein (CRP) >150mg/dl and polyethylene exchange), were also collected. Results. 30 patients had a successful DAIR outcome (60%). Age and sex do not act as risk factors [OR 0.7 (0.2-2.6) and OR 0.4 (0.1-1.3)]. Neither do COPD [OR 3.3 (0.5-2.0), p=0.2]; RA [OR 0.8 (0.2-3.1), p=0.7]; CRP value [3.2 (0.9-11.2), p=0.06]; and polyethylene exchange [OR 0.4 (0.1-2.5), p= 0.3]. Thirty-five blood cultures (70%) were obtained before surgery (20 SG and 15 FG). Nine of the obtained blood cultures were positive (25.7%), being 7 from FG (46.7%) [OR 7.6 (1.3-4.8), p=0.02]. A logistic regression was performed where positive blood cultures were the only significant variable to predict DAIR failure (OR 12, 95% CI 1.1−18, p=0.049), after adjusting for all CRIME80 variables. Skin and soft tissue origin was described in 5 of the nine positive blood cultures (55.6%). Cardiovascular system was the second most common spread (22.2%), and then followed by urogenital and digestive tract. The most common microorganism in FG was Staphylococcus aureus (57.1%) [OR 6.4 (0.2-18.0), p=0.2]. Conclusions. Positive blood cultures may be another risk factor for DAIR failure. This can be important in diagnosis and it may be taken into account in antibiotic and surgical treatment strategies

Aim. The aim of this study was to investigate the clinical relevance of an isolated positive sonication fluid culture (SFC) in patients who underwent revision surgery of a prosthetic joint. We hypothesized that cases with a positive SFC have a higher rate of infection and prosthesis failure during follow-up compared to controls with a negative SFC. Method. This retrospective multicentre observational study was performed within the European Study Group of Implant-Associated Infections (ESGIAI). All patients who underwent revision surgery of a prosthetic joint between 2013 and 2019 and had a minimum follow-up of 1 year were included. Patients with positive tissue cultures or synovial fluid cultures were excluded from the study. Results. 95 cases (positive SFC) and 201 controls (negative SFC) were included. There was no difference in infection and prosthesis failure during follow-up between both groups. When solely analysing patients that were not treated with antibiotics, 16% of the cases had an infection during follow-up versus 5% of the controls (P 0.046). Conclusions. Withholding antimicrobial treatment in patients with an isolated positive SFC is associated with a higher reinfection rate. Antimicrobial treatment should be considered in isolated positive SFC, especially in case of high virulent pathogens

Aims. The outcomes of patients with unexpected positive cultures (UPCs) during revision total hip arthroplasty (THA) and total knee arthroplasty (TKA) remain unknown. The objectives of this study were to establish the prevalence and infection-free implant survival in UPCs during presumed aseptic single-stage revision THA and TKA at mid-term follow-up. Methods. This study included 297 patients undergoing presumed aseptic single-stage revision THA or TKA at a single treatment centre. All patients with at least three UPCs obtained during revision surgery were treated with minimum three months of oral antibiotics following revision surgery. The prevalence of UPCs and causative microorganisms, the recurrence of periprosthetic joint infections (PJIs), and the infection-free implant survival were established at minimum five years’ follow-up (5.1 to 12.3). Results. Of the 297 patients undergoing aseptic revisions, 37 (12.5%) had at least three UPCs obtained during surgery. The UPC cohort included 23 males (62.2%) and 14 females (37.8%), with a mean age of 71.2 years (47 to 82). Comorbidities included smoking (56.8%), hypertension (48.6%), diabetes mellitus (27.0%), and chronic renal impairment (13.5%). The causative microorganisms included Staphylococcus epidermidis (49.6%), Bacillus species (18.9%), Micrococcus species (16.2%), and Cutibacterium acnes (16.2%). None of the study patients with UPCs developed further PJIs or required further surgical intervention during follow-up. Conclusion. The prevalence of UPCs during presumed aseptic revision THA and TKA was 12.5%. The most common causative microorganisms were of low virulence, and included S. epidermidis, Bacillus species, Micrococcus species, and C. acnes. Microorganism-specific antibiotic treatment for minimum three months’ duration of UPCs in presumed aseptic revision arthroplasty was associated with excellent infection-free implant survival at mid-term follow-up. Cite this article: Bone Jt Open 2024;5(10):832–836

Introduction. Cartilage comprises chondrocytes and extracellular matrix. The matrix contains different collagens, proteoglycans, and growth factors produced by chondroprogenitor cells that differentiate from proliferating to hypertrophic chondrocytes. In vitro chondrocyte growth is challenging due to differences in behaviour between 2D and 3D cultures. Our aim is to establish a murine 3D spheroid culture method using chondrocytes to study the complex interaction of cells on the chondro-osseous border during enchondral ossification. Method. Primary chondrocytes were isolated from the knee of WT new-born mice and used to form 10,000 cell number spheroids. We used the ATDC5-chondrocyte cell line as an alternative cell type. Spheroids were observed for 7, 14, and 21 days before embedding in paraffin for slicing. Alcian blue staining was performed to identify proteoglycan positive areas to prove the formation of extracellular matrix in spheroids. Collagen type 2, and Collagen type X expression were analyzed via quantitative real-time PCR and immunohistochemistry. Result. Alcian blue staining showed increasing matrix formation from day 7 to day 14 and proliferative chondrocytes at early time points. Both cell types showed increasing mRNA expression of Collagen type 2 from day 7 to day 21. Collagen type X positive staining starting from day 14 on confirmed the development of hypertrophic stage of chondrocytes. ATDC5 cells exhibited a slower progression in chondrogenic differentiation compared to primary chondrocytes. Conclusion. In chondrocyte spheroids, we observed proceeding differentiation of chondrocytes reaching hypertrophic phase. Primary chondrocytes showed faster development than ATDC5 cell line. Overall, spheroid culture of chondrocytes could be a good basis to study the interaction of different cells types of the chondro-osseous border by combination of chondrocytes with e.g., endothelial cells and osteoblasts within the spheroid. Those organoid cultures might also help to reduce animal experiments in the future, by mimicking complex regeneration procedures like bone growth or fracture healing. DFG(German Research Foundation)

Aim. Septic arthritis is a painful infection of articular joints that is typically treated by irrigation & debridement along with antibiotic therapy. There is debate amongst the medical community whether antibiotic administration should be delayed until fluid cultures have been taken to improve culture yield. However, delaying antibiotics can also have negative consequences, including joint destruction and sepsis. Therefore, the purposes of this study were to determine: 1) whether delayed antibiotic treatment affects culture yield and prognosis and 2) if the culture yield of patients treated for septic arthritis differs for hip, knee, and shoulder based on timing of antibiotic administration. Method. A retrospective analysis was conducted on 111 patients with septic arthritis of the hip, knee, or shoulder admitted from 3/2016 to 11/2018. In patients with multiple septic joints, each joint was analyzed individually (n=122). Diagnosis was determined by the treatment of irrigation & debridement and/or a positive culture. Patients without all intervention times recorded or with periprosthetic joint infection were excluded. Demographics, laboratory tests, culture results, and intervention times were obtained through chart review. Patients were grouped based on antibiotic therapy timing: >24 hours prior to arthrocentesis (Group 1), between 24 hours and 1 hour prior (Group 2), and 1 hour prior to post-arthrocentesis (Group 3). Analysis was conducted using chi-squared tests. Results. The mean age of each group were similar: Group 1 (n=38) 55.7 years, Group 2 (n=20) 57.2 years, and Group 3 (n=64) 54.8 years. No difference was observed in culture sensitivity between groups (p=0.825) with 71.1% (27/38) positive cultures in Group 1, 75% (15/20) in Group 2, and 76.6% (49/64) in Group 3. Similarly, frequency of related readmissions within 90 days (p=0.863) did not significantly vary: 26.3% (10/38) in Group 1, 20% (4/20) in Group 2, and 25% (16/64) in Group 3. Additionally, there were no significant differences in culture sensitivity in the knee (p=0.618; Groups: 87.5%, 75%, 70.6%), shoulder (p=0.517; Groups: 77.8%, 66.7%, 90%), and hip (p=0.362; Groups: 61.9%, 80%, 80%). Conclusions. Culture sensitivities and rates of readmission were similar for all patients regardless of antibiotic administration timing. These results suggest that antibiotic administration should not be delayed in septic arthritis to improve culture yield. However, the data does not suggest that early antibiotic administration will result in better clinical outcomes by lowering readmission rates. Further research is needed to better determine the clinical benefits that early administration of antibiotics may have on patient outcomes

Previous research has shown catabolic cell signalling induced by TNF-α and IL-1β within intervertebral (IVD) cells. However, these studies have investigated this in 2D monolayer cultures, and under hyper-physiological doses. Thus, we aim to revisit the catabolic responses of bovine IVD cells in vitro in 3D culture under increasing doses of TNF-α or IL-1β stimulation at three different timepoints. Primary bovine nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated and expanded for two weeks. Subsequently, NP and AF cells were encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks for phenotype recovery. Re-differentiated cells were stimulated with 0.1, 1 and 10 ng/ml TNF-α or with 0.01, 0.1 and 10 ng/ml IL-1β for one week. Beads were collected on the stimulation day (Day 0) and on Day 1 and 7 after stimulation. A dose-dependent upregulation of catabolic markers was observed in both cell types after one day of TNF-α or IL-1β stimulation. 10 ng/ml TNF-α stimulation induced a significant upregulation (p<0.05) of ADAMTS4, MMP3 and MMP13 in AF cells after one day of stimulation. Similarly, MMP3 upregulation showed a strong trend (p=0.0643) in NP cells. However, no effects on expression were seen after seven days. In addition, no significant difference between treatments in COL2, COL1 and ACAN expression was observed, and cell viability was not reduced at any time point, regardless of the treatment. We demonstrate a dose-dependent upregulation of catabolic markers in NP and AF cells under TNF-α or IL-1β stimulation, with a significant upregulation of ADAMTS4, MMP3 and MMP13 genes in AF cells after one day of treatment. Notably, after seven days of treatment, the dose-dependent effects were no longer observed possibly due to an adaptation mechanism of IVD cells to counter the metabolic shift

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

Poor tendon repair is an unsolved issue in clinical practice, due to complex tendon structure. Tendon stem/progenitor cells (TSPCs) play key roles in homeostasis, regeneration, and inflammation regulation in acute tendon injuries, and rely on TGF-β signaling for recruitment into degenerative tendons. In this study, we aimed to develop an in vitro model for tenogenesis adopting a dynamic culture of a fibrin 3D scaffold, bioengineered with human TSPCs collected from both healthy and tendinopathic surgery explants (Review Board prot./SCCE n.151, 29 October 2020). 3D culture was maintained for 21 days under perfusion provided by a custom-made bioreactor, in a medium supplemented with hTGF-β1 at 20 ng/mL. The data collected suggested that the 3D in vitro model well supported survival of both pathological and healthy cells, and that hTGF-β signaling, coupled to a dynamic environment, promoted differentiation events. However, pathological hTSPCs showed a different expression pattern of tendon-related genes throughout the culture and an impaired balance of pro-inflammatory and anti-inflammatory cytokines, compared to healthy hTSPCs, as indicated by qRT-PCT and immunofluorescence analyses. Additionally, the expression of both tenogenic and cytokine genes in hTSPCs was influenced by hTGF-β1, indicating that the environment assembled was suitable for studying tendon stem cells differentiation. The study offers insights into the use of 3D cultures of hTSPCs as an in vitro model for investigating their behavior during tenogenic events and opens perspectives for following the potential impact on resident stem cells during regeneration and healing events

Meniscus tears have been treated using partial meniscectomy to relieve pain in patients, although this leads to the onset of early osteoarthritis (OA). Cell-based therapies can help preserve the meniscus, although the presence of inflammatory cytokines compromises clinical outcomes. Anti-inflammatory drugs (e.g. celecoxib), can help to reduce pain in patients and in vitro studies suggest a beneficial effect on cytokine inhibited matrix content. Previously, we have demonstrated that the inhibitory effects of IL-1β can be countered by culture under low oxygen tension or physioxia. The present study sought to understand whether physioxia, celecoxib or combined application can counter the inhibitory effects IL-1β inhibited meniscus cells. Human avascular and vascular meniscus cells (n =3) were isolated and expanded under 20% (hyperoxia) or 2% (physioxia) oxygen. Cells were seeded into collagen scaffolds (Geistlich, Wolhusen) and cultured for 28 days either in the presence of 0.1ng/mL IL-1β, 5µg/mL celecoxib or both under their expansion oxygen conditions. Histological (DMMB, collagen I and collagen II immunostaining), GAG content and gene expression analysis was evaluated for the scaffolds. Under hyperoxia, meniscus cells showed a significant reduction in GAG content in the presence of IL-1β (*p < 0.05). Celecoxib alone did not significantly increase GAG content in IL-1β treated cultures. In contrast, physioxic culture showed a donor dependent increase in GAG content in control, IL-1β and celecoxib treated cultures with corresponding histological staining correlating with these results. Additionally, gene expression showed an upregulation in COL1A1, COL2A1 and ACAN and a downregulation in MMP13 and ADAMTS5 under physioxia for all experimental groups. Physioxia alone had a stronger effect in countering the inhibitory effects of IL-1β treated meniscus cells than celecoxib under hyperoxia. Preconditioning meniscus cells under physioxia prior to implantation has the potential to improve clinical outcomes for cell-based therapies of the meniscus

The novel, highly-sensitive and non-destructive method for the quantification of the osteogenic potential of bone marrow mesenchymal stem cells (BM-MSCs), by the evaluation of its hydroxyapatite (HA), in vitro is 99mTc-HDP-Labelling. 99mTc-HDP (tracer) binds rapidly to HA and this uptake can be visualized and quantified. This study was performed to evaluate if this method is suitable to perform a real-time assessment during an ongoing cell culture and if the radioactive tracer may influence the cells and their ability to differentiate. BM-MSCs (n=3) were cultivated in 35mm-dishes. Groups 1 and 3 received DMEM-LG based osteogenic media while Groups 2 und 4 were non-osteogenic controls. Groups 1 and 2 (multi-labelling) were incubated with 5 MBq 99mTc-HDP for 30min on day 7 (d7) and the bound activity was measured using an activimeter. Subsequently the cell-culture was continued and again labelled with 99mTc-HDP on day 14 and 21 (d14, d21). Groups 3 and 4 (single labelling), cultivation of the respective triplicates, ended on day 7, 14 and 21 (d7, d14, d21) followed by 99mTc-HDP-Labelling. Statistical analysis using one-factor ANOVA (p<0.05). Absolute tracer uptake increased steadily in both osteogenic groups: 1 (d7: 0.315; d14: 1.093; d21: 3.283 MBq) and 3 (d7: 0.208; d14: 0.822; d: 212.437 MBq) and was significantly higher than in the corresponding non-osteogenic control-group (Group 2 and 4) at all timepoints. (p<0.001). No significant negative effect of the radioactive tracer could be revealed in group 1 (multi radioactive labelling on d7, d14, d21) compared to Group 3 (singe labelling). The 99mTc-Uptake of groups 2 and 4 was not significantly different at any time. Our data show that the repeated exposition to 99mTc-HDP has no negative influence on the osteogenic differentiation potential of BM-MSCs. Therefore, the method is capable of determining the amount of HA during an ongoing cell culture

Autografts containing bone marrow (BM) are current gold standard in the treatment of critical size bone defects, delayed union and bone nonunion defects. Although reaching unprecedented healing rates in bone reconstruction, the mode of action and cell-cell interactions of bone marrow mononuclear cell (BM-MNC) populations have not yet been described. BM-MNCs consist of a heterogeneous mixture of hematopoetic and non-hematopoetic lineage fractions. Cell culture in a 3D environment is necessary to reflect on the complex mix of these adherend and non-adherend cells in a physiologically relevant context. Therefore, the main aim of this approach was to establish conditions for a stable 3D BM-MNC culture to assess cellular responses on fracture healing strategies. BM samples were obtained from residual material after surgery with positive ethical vote and informed consent of the patients. BM-MNCs were isolated by density gradient centrifugation, and cellular composition was determined by flow cytometry to obtain unbiased data sets on contained cell populations. Collagen from rat tail and human fibrin was used to facilitate a 3D culture environment for the BM-MNCs over a period of three days. Effects on cellular composition that could improve the regenerative potential of BM-MNCs within the BM autograft were assessed using flow cytometry. Cell-cell-interactions were visualized using confocal microscopy over a period of 24 hours. Cell localization and interaction partners were characterized using immunofluorescence labeled paraffin sectioning. Main BM-MNC populations like Monocytes, Macrophages, T cells and endothelial progenitor cells were determined and could be conserved in 3D culture over a period of three days. The 3D cultures will be further treated with already clinically available reagents that lead to effects even within a short-term exposure to stimulate angiogenic, osteogenic or immunomodulatory properties. These measures will help to ease the translation from “bench to bedside” into an intraoperative protocol in the end

Novel biomaterials are being developed and studied, intended to be applied as bone graft substitute materials. Typically, these materials are being tested in in vitro setups, where among others their cytotoxicity and alkaline phosphatase activity (as a marker for osteoblastic differentiation) are being evaluated. However, it has been reported that in vitro tests correlate poorly with in vivo results and therefore many promising biomaterials may not reach the clinic as a bone graft substitute product. One of the reasons for the poor correlation, may be the minimal complexity of the in vitro tests, as compared to the in vivo environment. Ex vivo models, mimicking the natural tissue environment whilst maintaining control of culture parameters, may be a promising alternative to assess biomaterials for bone formation. Assess the possibility of an ex vivo culture platform to test biomaterials on their potential to stimulate new bone formation. Osteochondral plugs (cylinders n=10, Ø 10 mm, height 15 mm) were drilled from fresh porcine knees, from the slaughterhouse. A bone defect (Ø 6 mm) was created and which was filled with a biomaterial graft (S53P4 bioactive glass (n=3); collagen sponges loaded with BMP-2 (n=3, as positive control)) or kept empty (n=4). The explants were cultured in custom-made two-chamber bioreactors for six weeks (LifeTec Group BV). Cartilage and bone were physically separated, similar to the in vivo situation, by a sealing ring. The two tissues were cultured in separate compartments, allowing for specific culture medium for each tissue. Medium was changed every 2–3 days and weekly micro computed tomography (µCT) images were obtained to longitudinally monitor the formation of new bone. An MTT assay was performed on half of the samples after six weeks of culture. The other samples were fixed for histology, to determine which cells were present after six weeks. The MTT metabolic assay showed that a number of cells in the bone were viable after six weeks. The further away from the border, the fewer living cells were observed. The cells in the cartilage also survived. No significant bone formation was observed with µCT in either of groups, even though abundant bone formation was expected in the BMP-2 group. Explanations of the negative results of the positive group might be that too few viable cells remain after six weeks, or that the cells that are still present are not able to form bone. No significant bone formation was observed in the bone defects in osteochondral explants that were cultured with, or without, biomaterials for six weeks. However, the platform showed that it is capable to successfully culture osteochondral explants for six weeks. Histology needs to be performed to evaluate which cells were present at the end of the culture and this will be compared to the cells present directly after drilling the explants

Aim. Periprosthetic joint infection (PJI) is one of the most devastating complications after joint replacement. It is associated with high morbidity and economic burden when misdiagnosed as an aseptic failure. Among all cases of PJI, up to 25% could yield negative cultures. Conversely, among cases of aseptic failures, up to 30% may actually be undiagnosed PJIs. In PJIs microbiological diagnosis is a key step for successful treatment. Sonication of the removed prosthesis is more sensitive than conventional periprosthetic-tissue culture, especially in patients who received antimicrobial therapy before surgery. This study aimed to compare the diagnostic value of classic sonication fluid cultures (SF-C) and sonication fluid incubation in blood culture bottle (SF-BCB). Method. Between 2016 and 2018 we analysed 160 revision procedures of joint arthroplasties. For each procedure, at least 5 microbiological and multiple histopathological samples were harvested, and explant sonication was performed which was further analysed by SF-C and SF-BCB. For SF-C classical cultivation of sonication fluid was performed. While for SF-BCB, 10 mL of sonication fluid was inoculated into aerobic and anaerobic lytic blood culture bottles. The definite diagnosis of PJI was based on the EBJIS definition. Results. Among 160 revisions, 59 PJIs were identified, 15 patients were treated with the debridement and implant retention, 7 patients with the one-stage and 35 with the two-stage exchange, remaining 2 were partial revisions. The sensitivity of SF-C and SF-BCB were 81.5% and 94.9%, respectively. The mismatch of microbe identification was observed in 5 cases. We observed positive SF-C while negative SF-BCB in 4 cases, among them having 2 positive histology. While 12 patients have negative SF-C and positive SF-BCB, among them 3 have positive and 6 negative histology. Among these 12 patients, typical low-grade microbes were identified in 9 cases (5 cases of C. acnes, 3 cases of S. epidermidis, and 1 case of S. capitis). Conclusions. The weakest point in all PJI diagnostic criteria is their sensitivity. SF-BCB demonstrates higher sensitivity in diagnosing PJI compared to SF-C. Therefore, it appears prudent to incorporate SF-BCB into the diagnostic protocol for all patients exhibiting either low-grade PJI symptoms or experiencing undiagnosed, presumably aseptic failures, where the likelihood of misdiagnosing infection is greatest

Aim. Septic arthritis (SA) is considered a medical emergency. The most common etiological agents are glucose consuming bacteria, so we evaluated the clinical utility of synovial fluid (SF) glucose levels and other biochemical parameters for supporting the diagnosis of the disease and their association with a positive bacteria culture and joint destruction. Methods. Adult patients with SA diagnose were enrolled prospectively between July 2018 and October 2019. As control group, adults with knee osteoarthritis, meniscus and/or knee ligaments lesions were enrolled. SF samples were obtained from the joints by arthrocentesis/arthrotomy. Microbiological analyses of SF were performed using Brucella broth blood culture flasks, samples were incubated at 37°C with 5% CO. 2. for 24 hours. Gram stain, chocolate and blood agar were used for the identification and growth of the bacteria. SF glucose levels, pH and leukocyte esterase were measured as biochemical parameters using a glucometer and colorimetric test strips. The Outerbridge classification was used for grading the osteochondral injury. Furthermore, blood samples were collected from patients and control subjects for determining glucose levels. Results. We included 8 subjects with knee ligaments lesions, 6 with meniscus lesions and 5 with osteoarthritis as control group, as well as 20 patients with SA diagnose. The mean age of the patients was 57.8 years with a 65% of male predominance. The most common affected joint was the knee (85%). SF culture was positive in 60% of the cases and the most common etiological agent was Staphylococcus aureus (58.3%). SF glucose levels from patients were lower than the controls (P=0.0018) and showed the lowest concentration in patients with a positive culture (P=0.0004). There was also a difference between blood and SF glucose concentration from the positive culture patients (P<0.0001). Leucocyte esterase presented the highest values in positive culture patients (P=<0.0001) and a more acidic pH was found compared to the control group (P<0.0001). Regarding the osteochondral injury, the lowest concentrations of SF glucose were found in patients with a higher grade in the classification (P = 0.0046). Conclusions. SF glucose and leukocyte esterase concentrations might be a quick and cheap useful parameter for the physician for distinguishing between bacterial infection and not infected joint. In addition, the lowest SF glucose levels might give information about the joint damage due to the disease

Aim. The primary objective is to evaluate the diagnostic performance of inoculating homogenized tissue and bone biopsies in blood culture bottles (BCB) for patients with (suspected) orthopaedic device-related infections. As secondary objective the time to positivity (TTP) of BCB and Wilkins-Chalgren broth (conventional method) will be evaluated. Method. Patients undergoing revision surgery due to suspected or proven fracture-related infection (FRI) or periprosthetic joint infection (PJI) according to respectively Consensus definition and EBJIS definition are included. 1,2. A minimal of three macroscopic infected/inflamed tissue/bone samples are collected in a container with saline and glass beads. 1.5 mL of the homogenized suspension is inoculated in BacT/ALERT FA and FN Plus bottles for 14 days. The remaining suspension is inoculated in Wilkins-Chalgren broth for 10 days and subcultured when cloudy or after 10 days. TTP is defined as the time until definite identification of the pathogen in the Laboratory Information System. Results. Up to now, 25 patients have been included, 11 (44%) had concordant results in BCB and the CM. In 11 patients cultures showed negative results for both methods. Three patients tested positive with BCB but remained negative with the same pathogen in CM. In the first patient, the CM failed to identify anaerobic bacteria (i.e. Fusobacterium nucleatum). In the second patient, three BCB were positive with Staphylococcus capitis. The third patient showed an infection with Escherichia coli, which was detected in all samples from the BCB, while all cultures obtained with the CM remained negative. A possible explanation for this discrepancy could be that this patient already received antibiotic therapy. BCB contain resins, which are capable of neutralizing antibiotic activity. Another case illustrating superiority of BCB involved an infection with Cutibacterium acnes, which showed positivity in six BCB, while only three were positive using the CM. We observed the shortest TTP with BCB. The median TTP of BCB was 32.0 hours (IQR 29.8) compared to a median TTP of 77.5 hours (IQR 107.6) when culturing with the CM. Contamination was seen in three patients with both methods, in eight patients contamination was only seen with the CM. For the remaining 14 patients no contamination was found. Conclusions. The results in this ongoing study indicate that the recovery of pathogens and TTP is better using BCB compared to CM. In addition, contamination occurs less frequently with the BCB method. Culturing tissue or bone biopsies in BCB seems a promising and faster detection method

Introduction and Objective. Low back pain (LBP) is a disorder strongly associated with intervertebral disc degeneration (IDD) with an important impact on the quality of life of affected people. To date, LBP treatment is based on conservative methods with the aim to reduce back pain without restoring the degenerative environment of the disc. The main cause of IDD is the drastic reduction of the proteoglycan content within the nucleus pulposus (NP), eventually leading to the loss of disc water content, micro-architecture, biochemical and mechanical properties. A promising approach for disc regeneration is represented by the transplantation of mesenchymal stromal cells (MSCs). The exact mechanism remains unknown. Growing evidence suggests that MSCs can influence cells and modulate cells’ behaviour by secreting a set of bioactive factors. MSCs secretome is composed of several molecules such as soluble protein, lipids, nucleic acids and extracellular vesicles (EVs) involved in inflammation, immunomodulation, cell survival and intercellular communication. The aim of this study was to evaluate the in vitro effects of MSCs secretome on human NP cells (hNPCs) in a 3D culture model with and without inflammatory stimulus. Materials and Methods. MSCs secretome was collected from bone marrow-MSCs (BM-MSCs) and adipose tissue-MSCs (ASCs) after centrifugation and obtained by culturing cells without fetal bovine serum (FBS) for 48 hours. hNPCs were isolated from surgical specimens through digestion with type II collagenase, culture expanded in vitro, encapsulated in alginate beads (three-dimensional culture system) and treated with growth medium (controls), BM-MSCs or ASCs secretome with or without interleukin-1 beta (IL-1b). After 7 days, total RNA was extracted and reverse-transcribed. Gene expression levels of catabolic and anabolic genes were analyzed through real time-polymerase chain reaction (qPCR). Cell proliferation and glycosaminoglycan (GAG) production was assessed by flow cytometry and 1,9-dimethylmethylene blue (DMMB), respectively. hNPCs in alginate beads were stained with Live/Dead assay and detected using confocal immunofluorescence microscopy. Data were analyzed using Graphpad prism 8 and expressed as mean ± S.D. One-way ANOVA analysis was used to compare differences among the groups under exam. Results. Our results reported an increase of hNPCs proliferation after treatment with both MSCs-secretomes. In detail, cell proliferation levels increased at 7 days after ASC-secretome (p ≤ 0,05) and BM-secretome (p ≥ 0,05) treatment compared to control. Live/dead staining showed that cell death was reduced by BM-secretome (p ≤ 0,05); in combined treatment of BM-secretome with IL1b 10ng/mL (p ≤ 0,05) at 7 days compared to control. There is not a significant difference between treated and untreated hNPCs’ GAG synthesis. In addition, gene expression levels resulted to be modulated by MSCs-secretomes under study compared to controls. Conclusions. Although the cell-therapy may be considered an attractive and safe option, MSCs require long and expensive processes. In conclusion, our experimental conditions supported as BM-MSCs and ASCs secretomes could represent cell-free alternative approaches in IDD, overcoming translational limits of cell therapy to the clinical practice

Objectives. Bone tissue engineering is one of the fastest growing branches in modern bioscience. New methods are being developed to achieve higher grades of mineral deposition by osteogenically inducted mesenchymal stem cells. In addition to well established monolayer cell culture models, 3D cell cultures for stem cell-based osteogenic differentiation have become increasingly attractive to promote in vivo bone formation. One of the main problems of scaffold-based osteogenic cell cultures is the difficulty in quantifying the amount of newly produced extracellular mineral deposition, as a marker for new bone formation, without destroying the scaffold. In recent studies, we were able to show that . 99m. Tc-methylene diphosphonate (. 99m. Tc-MDP), a gamma radiation-emitting radionuclide, can successfully be applied as a reliable quantitative marker for mineral deposition as this tracer binds with high affinity to newly produced hydroxyapatite (HA). Methods. Within the present study, we evaluated whether this promising new method, using . 99m. Tc-hydroxydiphosphonate (. 99m. Tc-HDP), can be used to quantify the amount of newly formed extracellular HA in a 3D cell culture model. Highly porous collagen type II scaffolds were seeded with 1 × 106 human mesenchymal stem cells (hMSCs; n = 6) and cultured for 21 days in osteogenic media (group A – osteogenic (OSM) group) and in parallel in standard media (group B – negative control (CNTRL) group). After incubation with . 99m. Tc-HDP, the tracer uptake, reflected by the amount of emitted gamma counts, was measured. Results. We saw a higher uptake (up to 15-fold) of the tracer in the OSM group A compared with the CNTRL group B. Statistical analysis of the results (Student`s t-test) revealed a significantly higher amount of emitted gamma counts in the OSM group (p = 0.048). Qualitative and semi-quantitative analysis by Alizarin Red staining confirmed the presence of extracellular HA deposition in the OSM group. Conclusion. Our data indicate that . 99m. Tc-HDP labelling is a promising tool to track and quantify non-destructive local HA deposition in 3D stem cell cultures. Cite this article: T. L. Grossner, U. Haberkorn, T. Gotterbarm. . 99m. Tc-Hydroxydiphosphonate quantification of extracellular matrix mineralization in 3D human mesenchymal stem cell cultures. Bone Joint Res 2019;8:333–341. doi: 10.1302/2046-3758.87.BJR-2017-0248.R1