Objectives. The objective of this study was to determine if combining variations in mixing technique of antibiotic-impregnated polymethylmethacrylate (PMMA) cement with low frequency ultrasound (LFUS) improves antibiotic elution during the initial high phase (Phase I) and subsequent low phase (Phase II) while not diminishing mechanical strength. Methods. Three batches of vancomycin-loaded PMMA were prepared with different mixing techniques: a standard technique; a delayed technique; and a control without antibiotic. Daily elution samples were analysed using flow injection analysis (FIA). Beginning in Phase II, samples from each mix group were selected randomly to undergo either five, 15, 45, or 0 minutes of LFUS treatment. Elution amounts between LFUS treatments were analysed. Following Phase II, compression testing was done to quantify strength. A-priorit-tests and univariate ANOVAs were used to compare elution and mechanical test results between the two mix groups and the control group. Results. The delayed technique showed a significant increase in elution on day one compared with the standard mix technique (p < 0.001). The transition point from Phase I to Phase II occurred on day ten. LFUS treatments significantly increased elution amounts for all groups above control. Delayed technique resulted in significantly higher elution amounts for the five-minute- (p = 0.004) and 45-minute- (p < 0.001) duration groups compared with standard technique. Additionally, the correlations between LFUS duration and total elution amount for both mix techniques were significant (p = 0.03). Both antibiotic-impregnated groups exhibited a significant decrease in offset yield stress compared with the control group (p < 0.001), however, their lower 95% confidence intervals were all above the 70 MPa limit defined by International Standards Organization (ISO) 5833-2 reference standard for acrylic bone cement. Conclusion. The combination of a delayed mix technique with LFUS treatments provides a reasonable means for increasing both short- and long-term antibiotic elution without affecting mechanical strength. Cite this article: Dr. T. McIff. Combination of modified mixing technique and low frequency ultrasound to control the elution profile of vancomycin-loaded acrylic bone cement. Bone Joint Res 2016;5:26–32. doi: 10.1302/2046-3758.52.2000412

Aims

This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation.

This edition of Cochrane Corner looks at some of the work published by the Cochrane Collaboration, covering pharmacological interventions for the prevention of bleeding in people undergoing definitive fixation or joint replacement for hip, pelvic, and long bone fractures; interventions for reducing red blood cell transfusion in adults undergoing hip fracture surgery: an overview of systematic reviews; and pharmacological treatments for low back pain in adults: an overview of Cochrane Reviews

Aims. Second-generation metal-on-metal (MoM) articulations in total hip arthroplasty (THA) were introduced in order to reduce wear-related complications. The current study reports on the serum cobalt levels and the clinical outcome at a minimum of 20 years following THA with a MoM (Metasul) or a ceramic-on-polyethylene (CoP) bearing. Methods. The present study provides an update of a previously published prospective randomized controlled study, evaluating the serum cobalt levels of a consecutive cohort of 100 patients following THA with a MoM or a CoP articulation. A total of 31 patients were available for clinical and radiological follow-up examination. After exclusion of 11 patients because of other cobalt-containing implants, 20 patients (MoM (n = 11); CoP (n = 9)) with a mean age of 69 years (42 to 97) were analyzed. Serum cobalt levels were compared to serum cobalt levels five years out of surgery. Results. The median cobalt concentration in the MoM group was 1.04 μg/l (interquartile range (IQR) 0.64 to 1.70) at a mean of 21 years (20 to 24) postoperatively and these values were similar (p = 0.799) to cobalt levels at five years. In the CoP control group, the median cobalt levels were below the detection limit (< 0.3 μg/l; median 0.15 μg/l, IQR 0.15 to 0.75) at 20 years. The mean Harris Hip Score was 91.4 points (61 to 100) in the MoM group and 92.8 points (63 to 100) in the CoP group. Conclusion. This study represents the longest follow-up series evaluating the serum cobalt levels after 28 mm head MoM bearing THA and shows that serum cobalt concentrations remain at low levels at a mean of 21 years (20 to 24) after implantation. Cite this article:Bone Joint Res. 2020;9(3):145–150

Objectives. The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy. Methods. Using tenocytes from normal Sprague-Dawley rats, cultured both in control and high glucose conditions, reactive oxygen species (ROS) production, cell proliferation, messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, interleukin-6 (IL-6), matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 and type I and III collagens were determined after 48 and 72 hours in vitro. In an in vivo study, using diabetic rats and controls, NOX1 and 4 expressions in Achilles tendon were also determined. Results. In tenocyte cultures grown under high glucose conditions, gene expressions of NOX1, MMP-2, TIMP-1 and -2 after 48 and 72 hours, NOX4 after 48 hours and IL-6, type III collagen and TIMP-2 after 72 hours were significantly higher than those in control cultures grown under control glucose conditions. Type I collagen expression was significantly lower after 72 hours. ROS accumulation was significantly higher after 48 hours, and cell proliferation after 48 and 72 hours was significantly lower in high glucose than in control glucose conditions. In the diabetic rat model, NOX1 expression within the Achilles tendon was also significantly increased. Conclusion. This study suggests that high glucose conditions upregulate the expression of mRNA for NOX1 and IL-6 and the production of ROS. Moreover, high glucose conditions induce an abnormal tendon matrix expression pattern of type I collagen and a decrease in the proliferation of rat tenocytes. Cite this article: Y. Ueda, A. Inui, Y. Mifune, R. Sakata, T. Muto, Y. Harada, F. Takase, T. Kataoka, T. Kokubu, R. Kuroda. The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo. Bone Joint Res 2018;7:362–372. DOI: 10.1302/2046-3758.75.BJR-2017-0126.R2

Aim. Osteoarthritis (OA) is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control the expression of genes and are likely to regulate the OA transcriptome. We performed integrative genomic analyses to define methylation-gene expression relationships in osteoarthritic cartilage. Patients and Methods. Genome-wide DNA methylation profiling of articular cartilage from five patients with OA of the knee and five healthy controls was conducted using the Illumina Infinium HumanMethylation450 BeadChip (Illumina, San Diego, California). Other independent genome-wide mRNA expression profiles of articular cartilage from three patients with OA and three healthy controls were obtained from the Gene Expression Omnibus (GEO) database. Integrative pathway enrichment analysis of DNA methylation and mRNA expression profiles was performed using integrated analysis of cross-platform microarray and pathway software. Gene ontology (GO) analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Results. We identified 1265 differentially methylated genes, of which 145 are associated with significant changes in gene expression, such as DLX5, NCOR2 and AXIN2 (all p-values of both DNA methylation and mRNA expression < 0.05). Pathway enrichment analysis identified 26 OA-associated pathways, such as mitogen-activated protein kinase (MAPK) signalling pathway (p = 6.25 × 10-4), phosphatidylinositol (PI) signalling system (p = 4.38 × 10-3), hypoxia-inducible factor 1 (HIF-1) signalling pathway (p = 8.63 × 10-3 pantothenate and coenzyme A (CoA) biosynthesis (p = 0.017), ErbB signalling pathway (p = 0.024), inositol phosphate (IP) metabolism (p = 0.025), and calcium signalling pathway (p = 0.032). Conclusion. We identified a group of genes and biological pathwayswhich were significantly different in both DNA methylation and mRNA expression profiles between patients with OA and controls. These results may provide new clues for clarifying the mechanisms involved in the development of OA. Cite this article: A. He, Y. Ning, Y. Wen, Y. Cai, K. Xu, Y. Cai, J. Han, L. Liu, Y. Du, X. Liang, P. Li, Q. Fan, J. Hao, X. Wang, X. Guo, T. Ma, F. Zhang. Use of integrative epigenetic and mRNA expression analyses to identify significantly changed genes and functional pathways in osteoarthritic cartilage. Bone Joint Res 2018;7:343–350. DOI: 10.1302/2046-3758.75.BJR-2017-0284.R1

Objectives. Bisphosphonates (BP) are the first-line treatment for preventing fragility fractures. However, concern regarding their efficacy is growing because bisphosphonate is associated with over-suppression of remodelling and accumulation of microcracks. While dual-energy X-ray absorptiometry (DXA) scanning may show a gain in bone density, the impact of this class of drug on mechanical properties remains unclear. We therefore sought to quantify the mechanical strength of bone treated with BP (oral alendronate), and correlate data with the microarchitecture and density of microcracks in comparison with untreated controls. Methods. Trabecular bone from hip fracture patients treated with BP (n = 10) was compared with naïve fractured (n = 14) and non-fractured controls (n = 6). Trabecular cores were synchrotron scanned and micro-CT scanned for microstructural analysis, including quantification of bone volume fraction, microarchitecture and microcracks. The specimens were then mechanically tested in compression. Results. BP bone was 28% lower in strength than untreated hip fracture bone, and 48% lower in strength than non-fractured control bone (4.6 MPa vs 6.4 MPa vs 8.9 MPa). BP-treated bone had 24% more microcracks than naïve fractured bone and 51% more than non-fractured control (8.12/cm. 2. vs 6.55/cm. 2. vs 5.25/cm. 2. ). BP and naïve fracture bone exhibited similar trabecular microarchitecture, with significantly lower bone volume fraction and connectivity than non-fractured controls. Conclusion. BP therapy had no detectable mechanical benefit in the specimens examined. Instead, its use was associated with substantially reduced bone strength. This low strength may be due to the greater accumulation of microcracks and a lack of any discernible improvement in bone volume or microarchitecture. This preliminary study suggests that the clinical impact of BP-induced microcrack accumulation may be significant. Cite this article: A. Jin, J. Cobb, U. Hansen, R. Bhattacharya, C. Reinhard, N. Vo, R. Atwood, J. Li, A. Karunaratne, C. Wiles, R. Abel. The effect of long-term bisphosphonate therapy on trabecular bone strength and microcrack density. Bone Joint Res 2017;6:602–609. DOI: 10.1302/2046-3758.610.BJR-2016-0321.R1

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

Objectives. The aim of this study was to investigate the effect of granulocyte-colony stimulating factor (G-CSF) on mesenchymal stem cell (MSC) proliferation in vitro and to determine whether pre-microfracture systemic administration of G-CSF (a bone marrow stimulant) could improve the quality of repaired tissue of a full-thickness cartilage defect in a rabbit model. Methods. MSCs from rabbits were cultured in a control medium and medium with G-CSF (low-dose: 4 μg, high-dose: 40 μg). At one, three, and five days after culturing, cells were counted. Differential potential of cultured cells were examined by stimulating them with a osteogenic, adipogenic and chondrogenic medium. A total of 30 rabbits were divided into three groups. The low-dose group (n = 10) received 10 μg/kg of G-CSF daily, the high-dose group (n = 10) received 50 μg/kg daily by subcutaneous injection for three days prior to creating cartilage defects. The control group (n = 10) was administered saline for three days. At 48 hours after the first injection, a 5.2 mm diameter cylindrical osteochondral defect was created in the femoral trochlea. At four and 12 weeks post-operatively, repaired tissue was evaluated macroscopically and microscopically. Results. The cell count in the low-dose G-CSF medium was significantly higher than that in the control medium. The differentiation potential of MSCs was preserved after culturing them with G-CSF. Macroscopically, defects were filled and surfaces were smoother in the G-CSF groups than in the control group at four weeks. At 12 weeks, the quality of repaired cartilage improved further, and defects were almost completely filled in all groups. Microscopically, at four weeks, defects were partially filled with hyaline-like cartilage in the G-CSF groups. At 12 weeks, defects were repaired with hyaline-like cartilage in all groups. Conclusions. G-CSF promoted proliferation of MSCs in vitro. The systemic administration of G-CSF promoted the repair of damaged cartilage possibly through increasing the number of MSCs in a rabbit model. Cite this article: T. Sasaki, R. Akagi, Y. Akatsu, T. Fukawa, H. Hoshi, Y. Yamamoto, T. Enomoto, Y. Sato, R. Nakagawa, K. Takahashi, S. Yamaguchi, T. Sasho. The effect of systemic administration of G-CSF on a full-thickness cartilage defect in a rabbit model MSC proliferation as presumed mechanism: G-CSF for cartilage repair. Bone Joint Res 2017;6:123–131. DOI: 10.1302/2046-3758.63.BJR-2016-0083

Aims. We aimed to evaluate the temperature around the nerve root during drilling of the lamina and to determine whether irrigation during drilling can reduce the chance of nerve root injury. Materials and Methods. Lumbar nerve roots were exposed to frictional heat by high-speed drilling of the lamina in a live rabbit model, with saline (room temperature (RT) or chilled saline) or without saline (control) irrigation. We measured temperatures surrounding the nerve root and made histological evaluations. Results. In the control group, the mean temperature around the nerve root was 52.0°C (38.0°C to 75.5°C) after 60 seconds of drilling, and nerve root injuries were found in one out of 13 (7.7%) immediately, three out of 14 (21.4%) at three days, and 11 out of 25 (44.0%) at seven days post-operatively. While the RT group showed a significantly lower temperature around the nerve root compared with the control group (mean 46.5°C; 34.5°C to 66.9°C, p < 0.001), RT saline failed to significantly reduce the incidence of nerve root injury (ten out of 26; 38.5%; odds ratio (OR) 0.96; 95% confidence interval (CI) 0.516 to 1.785; p = 0.563). However, chilled saline irrigation resulted in a significantly lower temperature than the control group (mean 39.0°C; 35.3°C to 52.3°C; p < 0.001) and a lower rate of nerve root injury (two out of 21; 9.5%, OR 0.13; 95% CI 0.02 to 0.703, p = 0.010). Conclusion. Frictional heat caused by a high-speed drill can cause histological nerve root injury. Chilled saline irrigation had a more prominent effect than RT in reducing the incidence of the thermal injury during extended drilling. Cite this article: Bone Joint J 2017;99-B:554–60

Objectives. To elucidate the effects of age on the expression levels of the receptor activator of the nuclear factor-κB ligand (RANKL) and osteoclasts in the periodontal ligament during orthodontic mechanical loading and post-orthodontic retention. Materials and Methods. The study included 20 male Sprague-Dawley rats, ten in the young group (aged four to five weeks) and ten in the adult group (aged 18 to 20 weeks). In each rat, the upper-left first molar was subjected to a seven-day orthodontic force loading followed by a seven-day retention period. The upper-right first molar served as a control. The amount of orthodontic tooth movement was measured after seven-day force application and seven-day post-orthodontic retention. The expression levels of RANKL and the tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were evaluated on day 7 (end of mechanical force loading) and day 14 (after seven days of post-orthodontic retention). Statistical analysis was performed using the t-test, and significance was set at p < 0.05. Results. There was no significant difference between the amount of tooth movement in the young group (0.96, standard deviation (. sd. ) 0.30mm) and that in the adult group (0.80mm, . sd. 0.28) (p > 0.05) after the seven-day force application. On the compression side, the expression of RANKL and TRAP-positive osteoclasts in both the young and the adult groups increased after the application of force for seven days, and then decreased at the end of the seven-day retention period. However, by the end of the period, the expression of RANKL on the compression side dropped to the control level in the young group (p > 0.05), while it was still higher than that on the control side in the adult group (p < 0.05). The expression of RANKL on the compression side did not show significant difference between the young and the adult groups after seven-day force application (p > 0.05), but it was significantly higher in the adult group than that in the young group after seven-day post-orthodontic retention (p < 0.05). Similarly, the decreasing trend of TRAP-positive osteoclasts during the retention period in the adult group was less obvious than that in the young group. Conclusions. The bone-resorptive activity in the young rats was more dynamic than that in the adult rats. The expression of RANKL and the number of osteoclasts in adult rats did not drop to the control level during the post-orthodontic retention period while RANKL expression and the number of osteoclasts in young rats had returned to the baseline. Cite this article: X. Li, M. Li, J. Lu, Y. Hu, L. Cui, D. Zhang, Y. Yang. Age-related effects on osteoclastic activities after orthodontic tooth movement. Bone Joint Res 2016;5:492–499. DOI: 10.1302/2046-3758.510.BJR-2016-0004.R2

Objectives. Platelet-rich fibrin matrix (PRFM) has been proved to enhance tenocyte proliferation but has mixed results when used during rotator cuff repair. The optimal PRFM preparation protocol should be determined before clinical application. To screen the best PRFM to each individual’s tenocytes effectively, small-diameter culture wells should be used to increase variables. The gelling effect of PRFM will occur when small-diameter culture wells are used. A co-culture device should be designed to avoid this effect. Methods. Tenocytes harvested during rotator cuff repair and blood from a healthy volunteer were used. Tenocytes were seeded in 96-, 24-, 12-, and six-well plates and co-culture devices. Appropriate volumes of PRFM, according to the surface area of each culture well, were treated with tenocytes for seven days. The co-culture device was designed to avoid the gelling effect that occurred in the small-diameter culture well. Cell proliferation was analyzed by water soluble tetrazolium-1 (WST-1) bioassay. Results. The relative quantification (condition/control) of WST-1 assay on day seven revealed a significant decrease in tenocyte proliferation in small-diameter culture wells (96 and 24 wells) due to the gelling effect. PRFM in large-diameter culture wells (12 and six wells) and co-culture systems induced a significant increase in tenocyte proliferation compared with the control group. The gelling effect of PRFM was avoided by the co-culture device. Conclusion. When PRFM and tenocytes are cultured in small-diameter culture wells, the gelling effect will occur and make screening of personalized best-fit PRFM difficult. This effect can be avoided with the co-culture device. Cite this article: C-H. Chiu, P. Chen, W-L. Yeh, A. C-Y. Chen, Y-S. Chan, K-Y. Hsu, K-F. Lei. The gelling effect of platelet-rich fibrin matrix when exposed to human tenocytes from the rotator cuff in small-diameter culture wells and the design of a co-culture device to overcome this phenomenon. Bone Joint Res 2019;8:216–223. DOI: 10.1302/2046-3758.85.BJR-2018-0258.R1