We have designed a prospective study to evaluate the usefulness of prolonged incubation of cultures from sonicated orthopaedic implants. During the study period 124 implants from 113 patients were processed (22 osteosynthetic implants, 46 hip prostheses, 54 knee prostheses, and two shoulder prostheses). Of these, 70 patients had clinical infection; 32 had received antibiotics at least seven days before removal of the implant. A total of 54 patients had sonicated samples that produced positive cultures (including four patients without infection). All of them were positive in the first seven days of incubation. No differences were found regarding previous antibiotic treatment when analysing colony counts or days of incubation in the case of a positive result. In our experience, extending incubation of the samples to 14 days does not add more positive results for sonicated orthopaedic implants (hip and knee prosthesis and osteosynthesis implants) compared with a conventional seven-day incubation period. Cite this article: Bone Joint J 2013;95-B:1001–6

Introduction and Objective. The rupture of the anterior cruciate ligament is a common sports injury and surgical reconstruction is often required to restore full function of the knee. Hamstring tendons are usually used as autografts. In addition to knee pain and stiffness, infections are feared complications after surgery. Incubation of the autograft in a vancomycin solution until implantation reduced the infection rate by about ten-fold. Recent studies showed no negative effect of vancomycin on the biomechanical properties of porcine tendons. A negative effect of high vancomycin concentrations on chondrocytes and osteoblast is reported, but the effect on tendon and tenocytes is not known. Materials and Methods. Rat Achilles tendons or isolated tenocytes were incubated with an increasing concentration of vancomycin (0 – 10 mg). Tendons were incubated for 0 – 40 minutes, while tenoyctes were incubated for 20 minutes followed by culturing for up to 7 days. Cell viability was assessed with PrestoBlue Assay and live/dead stain. The potential effect of vancomycin on the expression of tendon specific genes and extracellular matrix (ECM) genes was quantified. Possible structural changes of the tendon are analyzed. Results. Incubation of the tendons or tenocytes with 5 mg vancomycin for 20 minutes (clinical use) had no negative effects on the cell viability in the tendons or the isolated tenocytes, while incubation with the toxic control (ethanol) significantly reduced cell viability. Even twice the concentration and a longer incubation time had no negative effect on the cells in the tendons or the isolated cells. Vancyomycin did not affect the expression of Col1a1, Col3a1, and the tenocyte markers mohawk, scleraxis and tenomodulin. Conclusions. The results showed that clinical practice of wrapping the autograft in vancomycin did not impair the tenocyte viability. The expression of collagens and tenocyte markers was also not affected, neither in the incubated tendons nor in the isolated cells. This indicates that vancomycin had no effect on cell phenotype and the formation of the extracellular matrix, which, in addition to cell viability, is important for the performance of the autograft

This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Our hypothesis was that irisin would improve hNPC metabolism and proliferation. hNPCs were isolated from intervertebral discs and cultured in alginate beads. hNPCs were exposed to phosphate-buffered saline (PBS) or recombinant irisin (r-irisin) at 5, 10 and 25 ng/mL (n=4). Each experiment was performed in triplicate. Cell proliferation was assessed with trypan blue staining-automated cell counting and PicoGreen assay. Glycosaminoglycan (GAG) content was measured using the DMMB assay. Metabolic activity was assessed with the MTT assay and the Griess Reagent System. Gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and −3, aggrecan, interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 was measured by RT-PCR. MTT assay and ADAMTS-5, COL2, TIMP-1 and IL-1β gene expression were evaluated following incubation with 5, 10 and 25 ng/mL r-irisin for 24 hours and subsequent culture with 10 ng/ml IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with r-irisin). Irisin increased hNPC proliferation (p<0.001), metabolic activity (p<0.05), GAG content (p<0.01), as well as COL2 (p<0.01), aggrecan (p<0.05), TIMP-1 and −3 (p<0.01) gene expression, while decreasing MMP-13 (p<0.05) and IL-1β (p<0.001) mRNA levels. r-irisin pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p<0.001) and a decrease of IL-1β (p<0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-irisin increased hNPC metabolic activity (p<0.001), COL2 gene expression (p<0.05) and decreased IL-1β (p<0.05) and ADAMTS-5 levels (p<0.01). Irisin stimulates hNPC proliferation, metabolic activity, and anabolism by reducing IL-1β and catabolic enzyme expression while promoting matrix synthesis

Nitric oxide is a free radical which in vivo is solely produced during the conversion of the amino acid arginine into citrulline by nitric oxide synthase enzymes. Recently, the importance of nitric oxide on inflammation and bone metabolism has been investigated. However, the knowledge regarding possible in vitro effects of arginine supplementation on chondrogenic differentiation is limited. ATDC5, a cell line which is derived from mouse teratocarcinoma cells and which is characterized as chondrogenic cell line, were proliferated in Dulbecco's Modified Eagle Medium (DMEM)/F12 and subsequently differentiated in proliferation medium supplemented with insulin, transferrin and sodium-selenite and where arginine was added in four different concentrations (0, 7.5, 15 and 30 mM). Samples were harvested after 7 or 10 days and were stored at −80 °C for subsequent RNA isolation for qPCR analysis. To determine chondrogenic differentiation, Alcian Blue staining was performed to stain the proteoglycan aggrecan, which is secreted by differentiated ATDC5 cells. All measurements were performed in triplo. Alcian Blue staining showed a qualitative increase of proteoglycan aggrecan secretion in differentiated ATDC5 cells after treatment with 7 and 15 mM arginine, with additional increased expression of ColII, ColX, Bmp4 and Bmp6. Treatment with 30 mM arginine inhibited chondrogenic differentiation and expression of aforementioned genes, however, Cox-2 and Vegfa gene expression were increased in these samples. Bmp7 was not significantly expressed in any experimental condition. The obtained results are suggestive for a dose-dependent effect of arginine supplementation on chondrogenic differentiation and associated gene expression, with 7.5 and 15 mM as most optimal concentrations and implications for apoptosis after incubation with 30 mM arginine. A future recommendation would be to investigate the effects of citrulline in a similar experiment, as this shows even more promising results to enhance the nitric oxide metabolism in sepsis and bone healing

Little information exists when using cell viability assays to evaluate cells within whole tissue, particularly specific types such as the intervertebral disc (IVD). When comparing the reported methodologies and the protocols issued by manufacturers, the processing, working times, and dye concentrations vary significantly, making the assay's reproducibility a costly and time-consuming trial and error process. This study aims to develop a detailed step-by-step cell viability assay protocol for evaluating IVD tissue. IVDs were harvested from bovine tails (n=8) and processed at day 0 and after 7 days of culture. Nucleus pulposus (NP) and the annulus fibrosus (AF) 3 mm cuts were incubated at room temperature (26˚C) with a Viability/Cytotoxicity Kit containing Calcein AM and Ethidium Ethidium homodimer-1 for 2 hr, followed by flash freezing in liquid nitrogen. Thirty µm sections were placed in glass slides and sealed with nail varnish or Antifade Mounting Medium. The IVD tissue was imaged within the next 4h after freezing using an inverted confocal laser-scanning microscope equipped with 488 and 543 nm laser lines. Cell viability at day 0 (NP: 92±9.6 % and AF:80±14.0%) and day 7 (NP: 91±7.9% and AF:76±20%) was successfully maintained and evaluated. The incubation time required is dependent on the working temperatures and tissue thickness. The calcein-AM dye will not be retained in the cells for more than four hours. The specimen preparation and culturing protocol have demonstrated good cell viability at day 0 and after seven days of culture. Processing times and sample preparation play an essential role as the cell viability components in most kits hydrolyse or photobleach quickly. A step-by-step replicable protocol for evaluating the cell viability in IVD will facilitate the evaluation of cell and toxicity-related outcomes of biomechanical testing protocols and IVD regenerative therapies

Matrix metalloproteinase enzymes (MMPs) play a crucial role in the remodeling of articular cartilage, contributing also to osteoarthritis (OA) progression. The pericellular matrix (PCM) is a specialized space surrounding each chondrocyte, containing collagen type VI and perlecan. It acts as a transducer of biomechanical and biochemical signals for the chondrocyte. This study investigates the impact of MMP-2, -3, and -7 on the integrity and biomechanical characteristics of the PCM. Human articular cartilage explants (n=10 patients, ethical-nr.:674/2016BO2) were incubated with activated MMP-2, -3, or -7 as well as combinations of these enzymes. The structural degradative effect on the PCM was assessed by immunolabelling of the PCM's main components: collagen type VI and perlecan. Biomechanical properties of the PCM in form of the elastic moduli (EM) were determined by means of atomic force microscopy (AFM), using a spherical cantilever tip (2.5µm). MMPs disrupted the PCM-integrity, resulting in altered collagen type VI and perlecan structure and dispersed pericellular arrangement. A total of 3600 AFM-measurements revealed that incubation with single MMPs resulted in decreased PCM stiffness (p<0.001) when compared to the untreated group. The overall EM were reduced by ∼36% for all the 3 individual enzymes. The enzyme combinations altered the biomechanical properties at a comparable level (∼36%, p<0.001), except for MMP-2/-7 (p=0.202). MMP-induced changes in the PCM composition have a significant impact on the biomechanical properties of the PCM, similar to those observed in early OA. Each individual MMP was shown to be highly capable of selectively degrading the PCM microenvironment. The combination of MMP-2 and -7 showed a lower potency in reducing the PCM stiffness, suggesting a possible interplay between the two enzymes. Our study showed that MMP-2, -3, and -7 play a direct role in the functional and structural remodeling of the PCM. Acknowledgements: This work was supported by the Faculty of Medicine of the University of Tübingen (grant number.: 2650-0-0)

Introduction. Bone and joint infection (BJI) is often characterized by severe inflammation and progressive bone destruction. Osteocytes are the most numerous and long-lived bone cell type, and therefore represent a potentially important long-term reservoir of bacterial infection. Staphylococcus aureus is known to establish stable intracellular osteocytic infections, however, little is known about the less virulent yet second most prevalent BJI pathogen, S. epidermidis, associated with late-diagnosed, chronic BJI. Thus, this study sought to establish an in vitro model to study the infection characteristics of S. epidermidis in human osteocyte-like cells. Methods. SaOS2 cells (1 ×10. 4. cells/cm. 2. ) were grown to confluence either without differentiation, representing an osteoblast-like (OB) state (SaOS2-OB) or differentiated to an osteocyte-like stage (SaOS2-OY), using established methods. Four S. epidermidis strains used (ATCC-12228, ATCC-14990, ATCC-35984 and a clinical osteomyelitis strain RAH-SE1) were tested to be Lysostaphin-resistant, necessitating antibiotic (Levofloxacin) control of extracellular bacteria. Infection of host cells (OB or OY) was tested at three multiplicities of infection (MOI: 10, 100 and 1000). Extracellular bacteria were controlled by overnight incubation at a 10X minimum inhibitory concentration (MIC) of Levofloxacin and thereafter at 1XMIC. At each time point (days 1, 3, 5) viable intra- and extracellular bacteria were quantified. Result. All strains displayed similar intracellular infection and persistence capabilities in SaOS2-OB and SaOS2-OY. Independent of MOI, intracellular bacteria in SaOS2-OB decreased over time, becoming non-culturable by day 5. In contrast, SaOs2-OY displayed enhanced intracellular bacterial persistence at each time point. In the presence of increased Levofloxacin concentration (10XMIC), S. epidermidis could persist intracellularly for at least 14 days. Conclusion. This study showed for the first time that S. epidermidis can infect human osteocytes and persist intracellularly. Additionally, even a 10xMIC antibiotic concentration failed to eradicate intracellular bacteria, suggesting that persistence within osteocytes could contribute to treatment failure and establishment of chronic BJI

Abstract. Objective. The preparation of host degenerate cartilage for repair typically requires cutting and/or scraping to remove the damaged tissue. This can lead to mechanical injury and cartilage cell (chondrocytes) death, potentially limiting the integration of repair material. This study evaluated cell death at the site of cutting injury and determined whether raising the osmotic pressure (hyper-osmolarity) prior to injury could be chondroprotective. Methods. Ex vivo human femoral head cartilage was obtained from 13 patients (5 males and 8 females: 71.8 years old) with Ethical Permission and Patient consent. Cartilage wells were created using 3 or 5mm biopsy punches. Cell death at the wounded edge of the host cartilage and the edge of the extracted explants were assessed by quantifying the percentage of cell death (PCD) and measuring the width of the cell death zone at identified regions of interest (ROI) using the confocal laser scanning microscopy and image analysis software. To assess the chondroprotective effect of hyper-osmolarity, cartilage specimens were incubated in 340 or 600mOsm media, five minutes prior to injury to allow the chondrocytes to respond to the altered osmolarity. Wounded cartilage explants and cartilage wells were then cultured for a further 150 minutes following injury. Results. In 340mOsm media, the PCD around the 3mm cartilage wells was significantly less compared to the corresponding explants (20.05±10.24% vs 35.25±4.86%; P=0.0003). When using the 5mm biopsy punch, the PCD at the wound edges was significantly lower when compared to the 3mm cartilage wells (13.33±7.80% vs 20.05±10.24%; P=0.0121) at the same osmolarity. The width of the cell death zone for the well edges for both 3 and 5mm punches was significantly narrower when compared to their corresponding harvested cartilage explants in 340mOsm media (P<0.0001; P=0.0218, respectively). Exposing cartilage to raised osmolarity (600mOsm) prior to injury significantly reduced the PCD for cartilage wells produced by the 3mm biopsy punches (from 20.05±10.24% to 12.24±6.00%; P=0.0025). In addition, the zone of cell death was marginally reduced at the edges of the 5mm cartilage wells (19.25±15.78mm to 12.72±9.09mm; P=0.0499). Conclusions. The choice of biopsy punch and the osmolarity of the incubation medium prior to cartilage injury markedly affected the extent of chondrocyte death both at the edges of the cartilage wells and the explants. The smaller biopsy punch caused more chondrocyte death in the native cartilage wells compared to the larger punch, but this could be compensated for by the chondroprotective effect of raising the osmotic pressure. In general, there was less cell death at the wounded edges of the cartilage wells, compared to the explants. These results suggest that there is scope for further optimising the cutting implements used to create the cartilage wells and protecting chondrocytes by hyper-osmolarity in order to minimize cell death at cut edges and potentially enhance integration between cartilage repair material and host cartilage. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Introduction:. Exercise has showed to reduce pain and improve function in patients with discogenic low back pain (LBP). Although there is currently no biologic evidence that the intervertebral disc (IVD) can respond to physical exercise in humans, a recent study has shown that chronic running exercise is associated with increased IVD hydration and hypertrophy1. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types, including chondrocytes2. This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs). Our hypothesis is that irisin may improve hNPCs metabolism and proliferation. METHODS:. The hNPCs, isolated from discectomy surgical waste material (n = 5), were expanded and encapsulated in alginate beads. The hNPCs were treated with: i) only growth medium (control); ii) medium with recombinant irisin (r-IR) at different concentrations (5, 10 and 25 ng / mL); iii) medium with Interleukin-1β (IL1β); iv) medium with IL1β for 24 h and then with IL1β and r-IR; v) medium with r-IR for 24 h and then with r-IR and IL1 β. We evaluated proliferation (trypan blue and PicoGreen), metabolic activity (MTT), nitrite concentration (Griess), and expression levels of catabolic and anabolic genes via real-time polymerase chain reaction (qPCR). Each analysis was performed in triplicate for each donor and each experiment was performed three times. Data were expressed as mean ± S.D. One-way ANOVA was used for the groups under exam. RESULTS:. Irisin increased hNPCs proliferation (p < 0.001), metabolic activity at 10 ng/mL (p < 0.05), and GAG content at concentration of 10 ng/mL and 25 ng/mL (p < 0.01; p < 0.001, respectively). The production of nitrites, used as an indicator of cellular oxidative stress, was significantly decreased (p < 0.01). Gene expression levels compared to the control group increased for COL2A1 (p < 0.01), ACAN (p < 0.05), TIMP-1 and −3 (p < 0.01), while a decrease in mRNA levels of MMP-13 (p < 0.05) and IL1β (p < 0.001) was noticed. r-IR pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p < 0.001), as well as a decrease of IL-1β (p < 0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-IR led to an increment of hNPC metabolic activity (p < 0.001), COL2A1 gene expression (p < 0.05) and a reduction of IL-1β (p < 0.05) and ADAMTS-5 gene levels (p < 0.01). CONCLUSIONS:. The present study suggested that irisin may stimulate hNPCs proliferation, metabolic activity, and anabolism by reducing the expression of IL-1β and catabolic enzymes while promoting the synthesis of extracellular matrix components. Furthermore, this myokine was able to blunt the catabolic effect of in vitro inflammation. Our results indicate that irisin may be one of the mediators by which physical exercise and muscle tissues modulate IVD metabolism, thus suggesting the existence of a biological cross-talk mechanism between the muscle and the IVD

Prosthetic joint infections (PJI) occur infrequently, but they represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus and coagulase-negative S. epidermidis are the most commonly infecting agents associated with PJI. Nowadays, Gram-negative species like Escherichia coli and Pseudomonas aeruginosa are gaining relevance. The use of TiO2 conical nanotubular doped with fluorine and phosphorous (FP-cNT) surfaces is an interesting approach to prevent surface bacterial colonization during surgery and favouring the osseointegration. Despite of there are serum markers related with PJI, to date there is described no biomaterial-related marker that allows detecting PJI. Here we describe the adherence and the bactericidal effect of FP-cNT and its capacity of marking the non-fermenting bacteria that have been in contact with it by Al. This metal is delivered by FP-cNT in non-toxic concentrations (between 25 and 29 ng/mL). F-P-cNT layers on Ti6Al4V alloy were produced as described previously by our group. Ti6Al4V chemical polishing (CP) samples without nanostructure were used as control and produced as described previously. S. aureus 15981, S. epidermidis ATCC 35984, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 strains adherence study was performed using the protocol described by Kinnari et al. in 0.9% NaCl sterile saline with a 120 min incubation. After incubation, the samples were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Proportion of live and dead bacteria was calculated and studied by using ImageJ software. The experiments were performed in triplicate. The aluminum concentration was estimated in the supernatant after incubation and in the 0.22 µm filtered supernatant by atomic absorption in graphite furnace. The statistical data were analyzed by nonparametric Kruskal-Walis test and by pairwise comparisons using the nonparametric unilateral Wilcoxon test with a level of statistical significance of p<0.05. The values are cited as medians. Our results show that the bacterial adherence of all tested species significantly decreased on FP-cNT compared to CP except P. aeruginosa ATCC 27853: 19.8% for S. aureus 15981, 45.3% for S. epidermidis ATCC 35984 and 8.1% for E. coli ATCC 25922. The bacterial viability decreased 2-fold for S. aureus 15981, and 5-fold for S. epidemidis ATCC 35984, but increased 95% for P. aeruginosa ATCC 27853 and there no was variation for E. coli ATCC 25922 on FP-cNT compared to CP. Only supernatant P. aeruginosa ATCC 27853 shows significant Al detection after 120 min incubation (p<0.05). In summary, F-P cNT is a promising biomaterial that besides favoring osseointegration and potential usefulness as drug carrier, present bactericidal, non-stick ability (at least for staphylococci and E. coli) and is able to mark P. aeruginosa with Al, which could be potentially monitored in serum and urine in patients with PJI

Introduction. The annual incidence of fractures in the UK is almost 4%. Bone grafting procedures and segmental bone transport have been employed for bone tissue regeneration. However, their limited availability, donor site morbidity and increased cost mean that there is still a large requirement for alternative methods and there is considerable research into regeneration using bone morphogenetic proteins (BMPs). The aims of this study are to synthesise and combine BMP-2 with a novel nanocomposite and study its release. Materials and Methods. BMP-2 was synthesised using an E. coli expression system and purified. C2C12 cells were used to test its bioactivity using an alkaline phosphatase (ALP) assay. The modified solution evaporation method was used to fabricate 30% a-TCP/PLGA nanocomposite and it was characterized using SEM, TEM, TGA, XRD, EDX and particle size analysis. The release pattern of adsorbed BMP-2 was studied using an ELISA assay. Results. SEM suggests that there was a homogeneous distribution of a-TCP nanoparticles within the PLGA matrix. The concentration of BMP-2 adsorbed onto a-TCP/PLGA nanocomposites directly correlated with the incubation concentration of BMP-2. Approximately 10-15% of BMP-2 was adsorbed on to the discs, up to an incubation concentration of 25 μg/ml. At a higher incubation concentration (50 μg/ml), however, only 4% of the BMP-2 appears to have been adsorbed. The ALP activity shows that the BMP-2 was bioactive and successfully adsorbed onto the surface of the a-TCP/PLGA nanocomposite. A burst release pattern of BMP-2 was observed over 24h, being maximal at 2 h. Discussion. Increasing incubation concentrations of BMP-2 resulted in an increase of detected adsorbed BMP-2 on the discs, however this was not observed at the highest incubation concentration (50 μg/ml). As adsorption of BMP-2 onto the ground surface of the a-TCP/PLGA nanocomposite occurs primarily through electrostatic interactions between cationic BMP-2 and anionic a-TCP, this might reflect saturation in adsorption secondary to saturation of surface anionic a-TCP by BMP-2, or heterogeneity of the discs' content and/or surface area. Adsorbed BMP-2 was shown to have bioactivity which significantly increased with increasing incubation concentrations of BMP-2 and suggests this nanocomposite could have osteoinductive potential in-vivo. The burst pattern of BMP-2 release has been shown previously from BMP adsorbed onto mPCL/collagen/HA composite and this significantly increased the bone formation of critical-sized defects. Whilst a more sustained release profile of BMP-2 is generally considered desirable, this nanocomposite of a-TCP/PLGA has been shown to possess some osteoconductive and weak osteoinductive properties itself (unpublished). The addition of BMP-2 to the nanocomposite by adsorption results in an early burst release, which can promote the differentiation of mesenchymal cells into osteoblasts. The proliferation of these might then be sustained by the nanocomposite itself, without the need for sustained delivery of BMP-2. Conclusions. Bioactive BMP-2 was synthesised and combined with a-TCP/PLGA nanocomposite, producing a biodegradable and osteoinductive material which has potential for use in bone regeneration

Metal instrumentation (rods and screws) is used to stabilise the spine after trauma, malignancy or deformity. Approx 3% become infected often necessitating removal of metal. At surgery tissue samples and metal are removed for culture, but many clinical laboratories are not equipped to process metal or use simple culture methods. The causative bacteria exist as biofilms on the metal and they are often anaerobic and slow-growing, so conventional culture methods often fail to detect them. Also, they are common contaminants leading to diagnostic uncertainty. We have established a laboratory protocol to overcome these problems. Removed metalwork was sonicated and the sonicate centrifuged and the supernatant discarded. Quantitative aerobic and anaerobic culture of the resuspended pellet for 14 days and microscopy were carried out. Metalwork from 11 suspected infected cases was culture-positive (median 2857, 60–5000cfu/mL). Microscopy revealed an infection due to Candida albicans that would not have been detected otherwise. Bacteria were isolated from 8 of 10 non-infected cases (median 15, 0–35 cfu/mL). Conventionally processed samples failed to grow in 4 infected cases. (cfu/mL infected vs noninfected cases p=0.0093). Micro-organisms on spinal metalwork grow as biofilms and they require sonication to dislodge them. The causative bacteria are slow-growing and P acnes is anaerobic and requires prolonged incubation. S epidermidis and P acnes are common contaminants and quantitative culture helps to distinguish pathogens from contaminants, removing the diagnostic uncertainty that conventional methods give. Microscopy of the sonicate can reveal micro-organisms that fail to grow on culture. We recommend that sonication of metalwork, prolonged anaerobic incubation and quantitative culture be adopted to improve diagnostic clarity for spinal instrumentation infections

MiRNAs perform gene regulation that can target approximately 60% of human protein coding genes. Along with many cellular processes, miRNAs have been implicated in stem cell differentiation. Osterix (Osx), which is inhibited by mir-31, is required by MSCs for early osteoblast differentiation resulting in bone formation further downstream. We used antagomir functionalised gold nanoparticles (AuNPs) to block mir-31, which resulted in upregulation of Osx in pre-osteoblastic MG63 cells and human mesenchymal stem cells (MSCs). We used MG63 pre-osteoblastic cell line and human MSCs. Cytotoxicity of AuNPs was assessed by MTT, and cellular uptake of AuNPs was verified by TEM and ICP-MS. Osx RNA levels were determined by Fluidigm analysis and protein expression by In Cell Western analysis. Antagomir-functionalised AuNPs were incubated with cells for an initial 48 hours. (1) No cytotoxic effects were noted in either cell type. (2) Fluidigm analysis identified a varied gene response to antagomir delivery in both cell types, with MSCs recording a reduction of stem cell marker genes nestin, alcam, CD63, and CD44 at day 5 (indicating differentiation). (3) Osx protein levels were increased in both cell types after 48 hour incubation. (4) Downstream MSC analysis demonstrated accelerated osteogenesis at week 3 and 5 (verified by osteocalcin nodule formation) following 48 hour AuNP incubation. RNA analysis in both cell types suggested a shift away from proliferation towards osteoblastic differentiation. This was supported by Osx protein expression, which was increased in both MG63 cells and MSCs. Finally, an increase in the late osteogenic marker (osteocalcin) was verified at weeks 3 and 5 in MSCs after AuNP incubation for 48 hours. These results collectively infer successful delivery of mir-31 antagomirs, which are blocking mir-31-mediated suppression of Osx, resulting in an early increase in Osx, which accelerates MSC osteogenesis downstream

Objectives. Intra-articular injections of local anaesthetics (LA), glucocorticoids (GC), or hyaluronic acid (HA) are used to treat osteoarthritis (OA). Contrast agents (CA) are needed to prove successful intra-articular injection or aspiration, or to visualize articular structures dynamically during fluoroscopy. Tranexamic acid (TA) is used to control haemostasis and prevent excessive intra-articular bleeding. Despite their common usage, little is known about the cytotoxicity of common drugs injected into joints. Thus, the aim of our study was to investigate the effects of LA, GC, HA, CA, and TA on the viability of primary human chondrocytes and tenocytes in vitro. Methods. Human chondrocytes and tenocytes were cultured in a medium with three different drug dilutions (1:2; 1:10; 1:100). The following drugs were used to investigate cytotoxicity: lidocaine hydrochloride 1%; bupivacaine 0.5%; triamcinolone acetonide; dexamethasone 21-palmitate; TA; iodine contrast media; HA; and distilled water. Normal saline served as a control. After an incubation period of 24 hours, cell numbers and morphology were assessed. Results. Using LA or GC, especially triamcinolone acetonide, a dilution of 1:100 resulted in only a moderate reduction of viability, while a dilution of 1:10 showed significantly fewer cell counts. TA and CA reduced viability significantly at a dilution of 1:2. Higher dilutions did not affect viability. Notably, HA showed no effects of cytotoxicity in all drug dilutions. Conclusion. The toxicity of common intra-articular injectable drugs, assessed by cell viability, is mainly dependent on the dilution of the drug being tested. LA are particularly toxic, whereas HA did not affect cell viability. Cite this article: P. Busse, C. Vater, M. Stiehler, J. Nowotny, P. Kasten, H. Bretschneider, S. B. Goodman, M. Gelinsky, S. Zwingenberger. Cytotoxicity of drugs injected into joints in orthopaedics. Bone Joint Res 2019;8:41–48. DOI: 10.1302/2046-3758.82.BJR-2018-0099.R1

After the implantation of endoprotheses or osteosynthesis devices, implant-related infections are one of the major challenges. The surface of implants offers optimal conditions for the formation of a biofilm. Effective carrier systems for the delivery of adequate therapeutics would reduce the concentrations needed for successful treatment and improve cure rates. In cancer diagnosis and therapy, magnetic nanoparticles are concentrated in the target area by an external magnetic field. For orthopaedic applications, in vitro examinations showed that the addition of a magnetic implant in combination with an external magnetic field could increase the amount of MNPSNPs that accumulated in direct vicinity to the implant. The present examinations implemented an electromagnet to increase magnetic field strength and should show if the in vitro set up can be transferred to an in vivo mouse model. Additionally, the loading capacity of the MNPSNPs with enrofloxacin and its release kinetics were determined. Fluorescein-isothiocyanate (FITC) was covalently attached to MNPSNPs. For the in vitro set up, a peristaltic pump was used to establish a closed circuit which contained the MNPSNP dispersion and a magnetic platelet. After 5 minutes fluid samples were taken from the area around the magnetic platelet and analysed using a microplate reader. For the in vivo set up, a BALB/c mouse was implanted subcutaneously with the metallic platelet at the hind leg. The MNPSNP dispersion was injected into the tale vein and the hind leg of the mouse was placed immediately in a magnetic field of 1.9 T. After one week the implant was retrieved and examined by confocal laser scanning microscopy (CLSM). Liver, spleen and kidneys of the mouse were examined by magnetic resonance imaging (MRI). The loading capacity of the MNPs with enrofloxacin was examined by quantification of the enrofloxacin content in the incubation and washing solution after incubation. The release kinetics weres tested in PBS using UV/Vis-spectrometry. The solution in the remaining tube contained no detectable MNPs while the concentration in the vicinity of the platelet was 150 µg/ml. The mouse showed no clinical adverse effects. The CLSM examination revealed a considerable accumulation of the MNPs at the implant surface. MRI could show neither accumulated MNPs nor changes of organ structure. The loading capacity of the MNPs for enrofloxacin was approximately 95 µg/mg. A burst release of nearly a third of the loaded antibiotic occurred within the first 6 hours followed by a further steady release. Conclusion. Loading and release of enrofloxacin showed appropriate results. For future studies antibiotics like rifampicin or vancomycin will be implemented. This first in vivo trial demonstrated an implant-directed targeting of the MNPs and successfully transferred the principle into an in vivo model so that a main study with statistically significant animal numbers has started including histological examinations

Background. Staphylococcus aureus is a human pathogen involved in implant-related infections. In these diseases, biofilm production is the key pathogenic event, and it increases antibiotic resistance of the organism. Because this phenomenon, local delivery of antibiotics could allows reaching high concentrations in the infected tissue without the secondary effects linked to systemic administration. Here we report the use of a ceramic biomaterial (SBA-15) as a carrier of antibiotics in order to deliver them directly in the infected tissue. Material and methods. SBA-15 discs were loaded with vancomycin, rifampin and a combination of both according to the protocol described by Molina-Manso et al. Loaded discs were introduced in a 0.5 McFarland suspension of S. aureus 15981 and incubated during 6 and 24 hours in order to develop a biofilm. After incubation, samples were sonicated during 5 minutes and 1:10 serial dilutions were performed in order to count viable bacteria. All experiments were performed in triplicate. Results. A statistically significant decrease in the number of viable bacteria was detected for all antibiotics at 6 hours, and also for vancomycin and the combination. Rifampin showed an increase in the number of viable bacteria at 24 hours. No differences were detected between vancomycin and the combination of antibiotics. Conclusion. SBA-15 can carry antibiotics that have effect on bacterial biofilm. The use of rifampin alone showed a loss of the effect after 24 hours of incubation, probably due to the selection of resistant mutants that nullify the effect of the antibiotic. No differences have been detected between vancomycin alone and its combination with rifampin in this experiment

Cell sheets are manufactured from a high-density cell layer stabilized by its own freshly produced extracellular matrix (ECM). They could serve as versatile scaffolds for tissue repair. Unfortunately, their production often remains time-consuming requiring weeks of culturing. Ligament cell sheets are so far barely available. Regarding musculoskeletal tissues exposed to high repetitive biomechanical forces, the stability of cell sheets is insufficient. It could help to combine them with a biomechanical competent scaffold e.g. produced by an embroidering technique. Hence, we wanted to (1) develop a very rapid strategy to produce ACL ligamentocyte sheets within 24 h by using a thermoresponsive polymer surface, (2) use the sheets for scaffold seeding and (3) reflect the fibrocartilaginous transition zone of an ACL enthesis by combining sheets of ligamentocytes with chondrocytes or chondrogenic precursor cells as a strategy for directed seeding of two cell types on topologically different scaffold areas. Different cell numbers of lapine ACL ligamentocytes (L-ACLs), lapine articular chondrocytes (L-ACs) and human mesenchymal stromal cells (H-MSCs) were used for sheet formation. Experiments were performed with novel, self-assembled poly(glycidyl ether) (PGE) brushes based on random glycidyl methyl ether and ethyl glycidyl ether copolymers on polystyrene 12-well cell culture plates, which allow rapid sheet formation within 24 h. Uncoated plates served as controls. Temperature-triggered detachment was performed by 10 min incubation with PBS at ambient temperature before treatment with fresh warm PBS for 5 min at 37 degrees Celsius. Harvested cell sheets were transferred on polyglycolic acid (PGA) or embroidered poly-lactic acid / poly-co-caprolactone (PLA/P[LA-CL]) scaffolds, functionalized with collagen foam and fluorine gas treatment (prepared at the IPF in Dresden and the FILK in Freiberg). Cell distribution, growth, vitality and synthesis of ECM components were monitored up to 7 days. Cell numbers required for sheet preparation (3.9 cm2) depended strongly on the cell type (L-ACLs: 0.395 mio/cm2, L-AC: 0.342 mio/cm2, H-MSCs: 0.131 mio/cm2) and was highest for L-ACLs. The majority of cells survived sheet assembly, detachment, transfer onto the scaffolds and culturing. Cells migrated from the sheets into the scaffolds and spread through the scaffolds. L-ACLs and L-ACs produced ECM and maintained their phenotypes (type II collagen and sulfated glycosaminoglycans in L-AC sheets, decorin and tenascin C in L-ACL sheets). The presence and distribution of two cell types in scaffold cocultures (L-ACLs and H-MSCs) was proven by anti-human vimentin labeling. Hence, the PGE brush surface allows rapid formation (24 h) of cell sheets

Direct metal printed (DMP) porous iron implants possess promising mechanical and corrosion properties for various clinical application. Nevertheless, there is a requirement for better co-relation between in vitro and in vivo corrosion and biocompatibility behaviour of such biomaterials. Our present study evaluates absorption of porous iron implants under both static and dynamic conditions. Furthermore, this study characterizes their cytocompatibility using fibroblastic, osteogenic, endothelial and macrophagic cell types. In vitro degradation was performed statically and dynamically in a custom-built set-up placed under cell culture conditions (37 °C, 5% CO2 and 20% O2) for 28 days. The morphology and composition of the degradation products were analysed by scanning electron microscopy (SEM, JSM-IT100, JEOL). Iron implants before and after immersion were imaged by μCT (Quantum FX, Perkin Elmer, USA). Biocompatibility was also evaluated under static and dynamic in vitro culture conditions using L929, MG-63, HUVEC and RAW 264.7 cell lines. According to ISO 10993, cytocompatibility was evaluated directly using live/dead staining (Live and Dead Cell Assay kit, Abcam) in dual channel fluorescent optical imaging (FOI) and additionally quantified by flow cytometry. Furthermore, cytotoxicity was indirectly quantified using ISO conform extracts in proliferation assays. Strut size of DMP porous iron implants was 420 microns, with a porosity of 64% ± 0.2% as measured by micro-CT. After 28 days of physiological degradation in vitro, dynamically tested samples were covered with brownish degradation products. They revealed a 5.7- fold higher weight loss than statically tested samples, without significant changes in medium pH. Mechanical properties (E = 1600–1800 MPa) of these additively manufactured implants were still within the range of the values reported for trabecular bone, even after 28 days of biodegradation. Less than 25% cytotoxicity at 85% of the investigated time points was measured with L929 cells, while MG-63 and HUVEC cells showed 75% and 60% viability, respectively, after 24 h, with a decreasing trend with longer incubations. Cytotoxicity was analysed by two-way ANOVA and post-hoc Tukey's multiple comparisons test. Under dynamic culture conditions, live-dead staining and flow cytometric quantification showed a 2.8-fold and 5.7-fold increase in L929 and MG-63 cell survival rates, respectively, as compared to static conditions. Therefore, rationally designed and properly coated iron-based implants hold potential as a new generation of absorbable Orthopaedic implants

Summary. Prosthetic UHMWPE added with vitamin E and crosslinked UHMWPE are able to decrease significantly the adhesion of various bacterial and fungal strains limiting biomaterial associated infection and consequent implant failure. Introduction. Polyethylene abrasive and oxidative wear induces overtime in vivo a foreign-body response and consequently osteolysis, pain and need of implant revision. To solve these problems the orthopaedic research has been addressed to develop new biomaterials such as a crosslinked polyethylene with a higher molecular mass than standard Ultra High Molecular Weight Polyethylene (UHMWPE), and consequently a higher abrasive wear resistance and an antioxidant (vitamin E)-added UHMWPE to avoid oxidative wear. Nevertheless a feared complication of implant surgery is bacterial or fungal infection, initiated by microbial adhesion and biofilm formation, and related to the biomaterial surface characteristics. Staphylococci are the most common microorganisms causing biomaterial associated infection (BAI), followed by streptococci, Gram-negative bacilli and yeasts. With the aim to prevent BAI, the purpose of this study was to evaluate the adhesion of various microbial strains on different prosthetic materials with specific surface chemical characteristics, used in orthopaedic surgery. Methods. We compared the effects of vitamin E-added UHMWPE and crosslinked UHMWPE with that of standard GUR 1020 UHMWPE, upon the adhesion of ATCC biofilm-producing strains of Staphylococcus epidermidis, S. aureus, Escherichia coli and Candida albicans. After different incubation times the samples were sonicated to release the attached microorganisms and spread onto agar to quantify colony forming units (UFC)/ml. The biomaterials were physico-chemically characterised by means of scanning electron microscopy (SEM), water contact angle (CA) measurements and attenuated total reflectance (ATR)-fourier transform infrared (FTIR) spectroscopy, before and after adhesion assays. The experiments were assayed in triplicate and repeated a minimum of three times. A statistical analysis on results was conducted. Results. No significant difference of the surface roughness, CA and ATR-FTIR spectroscopy was found among the different biomaterials. After 3 and 7 h of incubation microbial adhesion rates were similar with no statistically relevant differences among the samples assayed. On the contrary, after 24 and 48 h of incubation a significantly (p<0.05 and p<0.01) different adhesion trend was achieved on the three biomaterials, highlighting a microbial adhesion significantly lower on vitamin E-added UHMWPE and crosslinked UHMWPE compared with that on standard UHMWPE. Discussion/Conclusion. Standard UHMWPE, vitamin E-added UHMWPE and crosslinked UHMWPE were chosen because of their diffusion in the clinical use. Previously we showed that vitamin E addition to the UHMWPE reduces the adhesive ability of various staphylococcal strains, compared with standard UHMWPE, and we correlated this results with its antioxidant properties. The results of this study indicate a quite similar significant reduction of bacterial and fungal adhesion on either vitamin E-added UHMWPE and crosslinked UHMWPE, if compared to standard UHMWPE at 48h. Further analysis on the chemical- physical characteristics of the UHMWPE surfaces and on their morphology are needed to explain the different adhesions

Prosthetic joint infections (PJI) occur infrequently, but they represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus are the most common infecting agents associated with acute PJI, and also appear in some cases of delayed PJI. 1. S. aureus biofilm development can be divided in two stages: adhesion and proliferation. 2. To avoid PJI bacterial adhesion has to be decreased. Hybrid organo-inorganic sol-gel coatings are proposed as a promising biomaterial improvement. 3. One of these compounds is a mixture of two organopolisiloxanes: 3-methacryloxypropyltrimethoxysilane (MAPTMS) and tetramethylorthosilicate (TMOS). The aim of this work was to evaluate bacterial adhesion on MAPTMS-TMOS coating compared to titanium parts made by powder metallurgy. MAPTMS-TMOS sol-gel coating was produced using a molar ratio of 1:2 (MAPTMS:TMOS) and dispersed in ethanol. The sol-gel was deposited by dip-coating on titanium parts made by powder metallurgy followed by a thermal treatment at 120 ºC for 30 minutes. 4. Titanium parts without sol-gel coating were used as control. S. aureus 15981 strain adherence study was performed using the protocol described by Kinnari et al. 5. with 90 min incubation. After incubation, the samples were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Proportion of total adhered, live and dead bacteria was calculated and studied by using ImageJ software. The experiments were performed in triplicate. The statistical data were analyzed by pairwise comparisons using the nonparametric Mann-Whitney test with a level of statistical significance of p<0.05. Values are cited and represented as medians. S. aureus 15981 adherence was 942-fold lower on MAPTMS-TMOS coating than on uncoated titanium. According with our results, MAPTMS-TMOS sol-gel coating is a promising antiadherent surface for S. aureus. More studies are necessary in order to evaluate this property with other species and strains