Aims. Osteoporosis is characterized by decreased trabecular bone volume, and microarchitectural deterioration in the medullary cavity. Interleukin-19 (IL-19), a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. The aim of our study was to investigate the effect of IL-19 on osteoporosis. Methods. Blood and femoral bone marrow suspension IL-19 levels were first measured in the lipopolysaccharide (LPS)-induced bone loss model. Small interfering RNA (siRNA) was applied to knock down IL-19 for further validation. Thereafter, osteoclast production was stimulated with IL-19 in combination with mouse macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The effect of IL-19 was subsequently evaluated using tartrate-resistant acid phosphatase (TRAP) staining and quantitative real-time polymerase chain reaction (RT-qPCR). The effect of IL-19 on osteoprotegerin (OPG) was then assessed using in vitro recombinant IL-19 treatment of primary osteoblasts and MLO-Y4 osteoblast cell line. Finally, transient transfection experiments and chromatin immunoprecipitation (ChIP) experiments were used to examine the exact mechanism of action. Results. In the LPS-induced bone loss mouse model, the levels of IL-19 in peripheral blood serum and femoral bone marrow suspension were significantly increased. The in vivo results indicated that global IL-19 deletion had no significant effect on RANKL content in the serum and bone marrow, but could increase the content of OPG in serum and femoral bone marrow, suggesting that IL-19 inhibits OPG expression in bone marrow mesenchymal stem cells (BMSCs) and thus increases bone resorption. Conclusion. IL-19 promotes bone resorption by suppressing OPG expression in BMSCs in a LPS-induced bone loss mouse model, which highlights the potential benefits and side effects of IL-19 for future clinical applications. Cite this article: Bone Joint Res 2023;12(11):691–701

Objectives. The osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) balance is of the utmost importance in fracture healing. The aim of this study was therefore to investigate the impact of nonosteogenic factors on OPG and RANKL levels. Methods. Serum obtained from 51 patients with long bone fractures was collected over 48 weeks. The OPG and serum sRANKL (soluble RANKL) concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Smoking habit, diabetes, and alcohol consumption were recorded. Results. Age and sex greatly influenced preoperative serum levels of OPG and sRANKL but differences were even more pronounced during fracture healing. Statistical significance was observed for overall serum levels of OPG (p = 0.001) and sRANKL (p < 0.001) in older men and women (age greater than 50 years). Interestingly, OPG levels increased over time in older women but decreased over time in older men. Conclusion. These data suggest that nonosteogenic factors, most significantly age and sex, have a major impact on sRANKL and OPG levels. Given the established association of OPG and sRANKL levels and nonunion, these findings seem to be of clinical relevance. Cite this article: J. Starlinger, G. Kaiser, A. Thomas, K. Sarahrudi. The impact of nonosteogenic factors on the expression of osteoprotegerin and RANKL during human fracture healing. Bone Joint Res 2019;8:349–356. DOI: 10.1302/2046-3758.87.BJR-2018-0116.R3

Osteoprotegerin (OPG) has been reported to be a novel protein that can suppress osteoclast differentiation and activation. This study examined the therapeutic effects of OPG on established periprosthetic osteolysis in a rat model. A bone cement prosthesis was inserted into the rat femur and polyethylene particles were continuously infused into the knee joint using an osmotic pump. After osteolysis was established in four weeks, rats were intravenously injected with vehicle (control group) or 1 mg/kg of OPG (OPG-1 group) or 10 mg/kg of OPG (OPG-10 group) every week until they were sacrificed at 8 weeks. Effects of direct injections of OPG into the knee joint were also investigated. Periprosthetic bone resorption was evaluated with bone mineral density and histomorphometric analysis of membranes composed of total area of interface membrane and inflammatory grading. Radiographs were evaluated for focal osteolysis with a blind manner. Periprosthetic bone resorption was significantly suppressed in OPG-10 group compared to the other groups (p < 0.05). Histomorphometric analyses showed less total area as well as less inflammatory grading of the interface membrane in OPG-10 group compared to other groups (p < 0.01). Radiographic osteolysis appeared to decrease in number in OPG-10 group. Direct injections of OPG into the knee joint appeared to be more effective compared to intravenous injections. The present study demonstrates that OPG has significantly restored the established periprosthetic osteolysis in our animal model. OPG may be a possible agent to retain the bone stock before revision surgery for failed prostheses. Conclusion: This study demonstrates that osteoprotegerin suppresses the progression of periprosthetic osteolysis and restores bone stock in a rat model

The Osteoprotegerin/RANK/RANKL system has been implicated in the biological cascade of events initiated by particulate wear debris and bacterial infection resulting in periprosthetic bone loss around loosened total hip arthroplasties (THA). Individual responses to such stimuli may be dictated by genetic variation and we have studied the effect of single nucleotide polymorphisms (SNPs) within these genes. We performed a case control study of the Osteoprotegerin, RANK and RANKL genes for possible association with deep sepsis or aseptic loosening. All patients included in the study were Caucasian and had had a cemented Charnley THA and polyethylene acetabular cup. Cases consisted of 91 patients with early aseptic loosening and 71 patients with microbiological evidence at surgery of deep infection. Controls consisted of 150 THAs that were clinically asymptomatic for over 10 years and demonstrated no radiographic features of aseptic loosening. DNA samples from all individuals were genotyped using Taqman allelic discrimination. The A allele (p<0.001) and homozygous genotype A/A (p<0.001) for the OPG-163 SNP were highly associated with aseptic failure. Additionally, the RANK-575 (C/T SNP) T allele (p=0.004) and T/T genotype (p=0.008) frequencies were associated with aseptic failure. No statistically significant relationship was found between aseptic loosening and the OPG- 245 or OPG-1181 SNPs. When the septic group was compared to controls, the frequency of the A allele (p<0.001) and homozygous genotype A/A (p<0.001) for the OPG-163 SNP were statistically significant. No statistically significant relationship was found between septic failure and the OPG- 245, OPG-1181 or RANK-575 SNPs. Aseptic loosening and possibly deep infection of THA may be under genetic influence to candidate susceptibility genes. SNP markers may serve as predictors of implant survival and aid pharmacogenomic prevention of THA failure

Abstract. Cranial cruciate ligament (CrCL) disease/rupture is a highly prevalent orthopaedic disease in dogs and common cause of pain, lameness, and secondary joint osteoarthritis (OA). Previous experiments investigating the role of glutamate receptors (GluR) in arthritic degeneration and pain revealed that OA biomarkers assessing early bone turnover and inflammation, including osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa-B ligand (RANKL) are more likely to be influenced by glutamate signalling. Moreover, interleukin-6 (IL-6) has a complex and potentially bi directional (beneficial and detrimental) effect, and it is a critical mediator of arthritic pain, OA progression and joint destruction. Objectives. 1) to recruit dogs undergoing CrCL disease/rupture surgery and obtain discarded synovial fluid (SF) and serum/plasma (ethics approval, RCVS:2017/14/Alves); 2) to quantify the biomarkers listed above in the SF and serum/plasma by enzyme linked immunosorbent assay (ELISA); 3) to assess radiographic OA at the time of surgery and correlate it with the biomarkers and clinical findings. Methods. Abnova, Abcam and AMSBIO ELISA kits were tested using a validation protocol relating the standard curve to a dilution series of SF and serum/plasma (1× to 1/50×), with and without SF hyaluronidase treatment to evaluate linearity, specificity and optimal dilutions. Validated ELISA kits were used to measure [IL-6], glutamate [glu], [RANKL] and [OPG] in SF and serum/plasma. For each dog, CrCL disease pre-operative lameness scores were graded as: (1) mild, (2) moderate (easily visible), (3) marked (encumbered), (4) non-weightbearing lameness. Blinded OA scoring was performed on radiographs [15–60, normal-severe OA]. Results. canine population (n=14) was of various breeds, aged between 2–10 years and weighing 17.1–45.5Kg; 42.86% male; 57.14% female; 83.33% males and 62.5% females were neutered. Lameness scores varied from 1 and 4 (average 2.07±1.12) and radiographic OA scores from 18 and 36 (average 27.86±5.11). Individual correlations in concentrations with respect to age, weight, lameness score (1–4) and OA scores (15–60) were tested. SF [glu] and lameness score were inversely correlated with higher levels of lameness corresponding to lower SF [glu] (P=0.0141). SF [RANKL] inversely correlated with weight (P=0.0045) and lameness score (P=0.0135), and serum [RANKL] inversely correlated with weight (P=0.0437). There was also a negative correlation between SF and serum [OPG] and weight (P=0.0165 and P=0.0208, respectively). No other significant correlations were detected. Overall, [glu] and [IL-6] are increased in SF compared to serum/plasma, by 12.84 and 1.28, respectively, whereas all the remaining biomarkers are higher (2–3 times) in the serum/plasma compared to SF. Principal component analysis (PCA) and Pearson correlation coefficient matrix [IL-6/glu/RANKL/OPG] (n=7) showed SF [IL-6] correlates with SF [glu] (rs=0.64) and strong positive correlations between SF/serum [RANKL] and SF/serum [OPG] (rs 0.68–0.96). Conclusions. Dogs with CrCL disease show an association between the bone remodelling markers RANKL and OPG, and the inflammatory cytokine IL-6, and to a lesser extent SF [glu]. Therapeutics targeting bone remodelling, IL-6 or GluR/[glu] may be of interest for the management of OA in dogs. 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

Background. Understanding of the pathogenetic mechanisms of non-union can not ignore bone remodelling and its cascade of processes at cellular and biochemical levels culminating in an incomplete structural and functional restoration of the damaged bone. Osteoprotegerin (OPG) is expressed by osteoblasts and functions as a decoy receptor that is able to control and to regulate osteoclastogenesis and therefore to prevent bone resorption. The objectives of our study were: to investigate OPG serum levels in shaft fractures non-union compared to controls; to assess the use of OPG as a marker for the early identification of fracture non-unions. Material and Methods. OPG serum levels were determined in 25 male patients (aged between 20–59 years, mean 35.44 ± 11.53) with a shaft fracture non-union at the time of minimum six months (mean 16.83 ± 10.87) since trauma and age matched with 25 male controls patients (aged 20–59 mean 35.44 ± 11.76) with a shaft fracture healed. All patients were correctly operated with different types of synthesis for complex fractures of a long bone (humerus, femur, tibia). Osteocalcin, bone isoenzyme of alkaline phosphatase and deoxypyridino-line (DPD) were also measured. Results. OPG levels were significantly higher in non-union cases compared to age matched controls (mean 10.17 ± 3.08 vs 8.54 ± 1.18 U/L; p=0.0084). DPD level was significantly higher in cases respect to controls (mean 7.9 ± 2.74 vs 3.8 ± 1.00 nmolDPD/mmol urinary creatinine excretion; p=0.0001). ROC analysis and the classification for probability cut-off show a very good negative predictive value (84%) for a cut-off of OPG 10 U/L, indicating that all patients having OPG lower than 10 U/L are probably free of non-union. Similarly, for an increase of 1 U/L of OPG there is an increase of probability of being a case of 92%. Higher OPG levels clearly carries a higher risk of non-union, thus indicating the usefulness of OPG evaluation in the follow-up of fractured patients. Larger groups will allow the estimation of the correct level of OPG threshold by age, which we are now able to estimate of about 8 U/L for young patients and 10 U/L for older ones in our population. Conclusion. Shaft fracture non-union may occur following appropriate osteosynthesis in consequence of a condition of altered bone osteoclastic activity. OPG could be directly involved in the pathogenesis of shaft fractures non-union and seems to be an accurate predictive marker in non-union evaluation

Introduction: Avascular necrosis (AVN) of the femoral head (FH) is a painful disorder of the hip that leads to hip collapse. The pathology of AVN involves ischemic events leading to the death of bone. Several biological substances participate in the balance between osteoclasts and osteoblasts, like osteoprotegerin, RANK and RANKL. The expression of these genes affects the maturation and function of osteoblasts and osteoclasts and determines the rate of bone remodeling. In this study, we investigate the expression of OPG, RANK and RANKL in osteonecrotic FHs derived from 44 patients with AVN. Methods and Materials: RNA and proteins were isolated from both necrotic and normal site of FHs of 44 patients diagnosed with AVN. Quantitative RT-PCR was performed for OPG, RANKL and RANK molecules by using the Light Cycler FastStart DNA Master Hybridization Probes kit (Roche). Western Blotting: 22 bone tissues were run on 4–12% NuPAGE gel (Invitrogen). Anti-OPG, anti-RANKL and anti-actin antibodies were used and membranes were immersed in ECL. Results: Quantitative RT-PCR: The mRNA levels of OPG were higher in the necrotic (median: 5.25) than the normal site (median: 4.19) of the FHs and their difference was statistically significant (p< 0.05). The expression of RANK and RANKL was significantly lower than that of OPG following a similar pattern between the necrotic and normal site. The mRNA values of RANK and RANKL were higher in the necrotic sites [necrotic median: 1.0/normal median: 0.85, necrotic median: 0.8, normal median: 0.3, respectively] than the normal, although they were not statistically significant. Western Blotting analysis: Normal sites from all FHs showed comparable OPG protein levels (median: 0.57) which were similar to those of normal (median: 0.63). Similar pattern to that of OPG was observed also for RANKL protein expression, where the median value for RANKL/F-actin ratio was 0.49 and 0.5 in normal and necrotic sites of FHs, respectively. Discussion: OPG, RANK and RANKL are key genes for maintaining the balance between osteoblasts and osteoclasts. Our results show marked differences in the expression of OPG between the necrotic and the normal sites of the FHs; however, mRNA levels of RANKL varied insignificantly between normal and necrotic part of FH while mRNA levels of RANK gene remain similar in both sides of FHs. In contrast, the production of OPG and RANKL at the protein level showed no remarkable divergence. This indicates that the expression and production pattern of RANK may play the key role in the maintenance of the balance between osteoblasts and osteoclasts in AVN

Osteoarthritis (OA) involves pathology in both articular cartilage and subchondral bone. The osteoprotegerin (OPG)/receptor activator of nuclear factor kappa beta ligand (RANK-L) balance is known to modulate bone turnover. We compared the bony changes in human total knee arthroplasty (TKA) and cadaveric controls. A qualitative increase in subchondral and ligamentous insertional bone mineral density was observed on micro-CT sections of TKA bone compared with cadaveric controls. In-situ hybridization of digoxygenase (DIG)-labelled OPG riboprobes showed selective uptake in osteoblasts but not osteocytes or osteoclasts in TKA bone. Those data suggested that the upregulation of OPG expression by osteoblasts may have precipitated the bony hypertrophy of end-stage OA. Altered joint mechanics produced by periarticular bone remodelling may precede the cartilage changes of osteoarthritis (OA). Recently, receptor activator of nuclear factor kappa beta (RANK), along with its soluble ligand (RANK-L), have been shown to induce both maturation and activation of bone-degrading osteoclasts. Activation of RANK on osteoclast cells by RANK-L is opposed by another soluble factor, osteoprotegerin (OPG). Thus RANK/OPG balance is important in regulating bone turnover. Here, we compared periarticular bone from patients with end-stage OA undergoing total knee arthroplasty (TKA) with those of cadaveric controls. We assessed bony, histological and molecular changes that are important in the pathogenesis of OA. Using in-situ hybridization, we found increased staining of digoxygenase (DIG)-labelled OPG in osteoblasts of TKA bone. A corresponding increase in subchondral and insertional bone was seen on micro-CT (μCT) sections from TKA bone in comparison with cadaveric controls. Those changes were accompanied by marked articular cartilage degeneration on histology. This study is the first of which we are aware that directly assessed the role of OPG in inducing the bony changes seen in human end-stage OA. We used μCT to compare corresponding samples qualitatively from TKA and cadaveric bone. Adjacent sections underwent hybridization of digoxygenase (DIG)-labelled OPG riboprobes to assess gene expression in situ. Finally, samples were stained and analysed for histology. Bony hypertrophy may be a result of overexpression of OPG that occurs as an important feature of OA pathophysiology. Funding: This work was supported by a grant from the Hip Hip Hooray Fund of the Canadian Orthopaedic Research Foundation (CORF) and the Wood Professorship in Joint Injury Research. There was no commercial funding for this research project

Objective: The clinical significance of biochemical bone markers in the diagnosis and severity of Osteoarthritis remains still unknown. The relationship between biochemical bone turnover markers and commonly recognizable radiographic features of knee and hip osteoarthritis remains unclear. Purpose: We evaluated the serum levels of Receptor Activator of Nuclear Factor-κB Ligand (RANKL), Bone-specific Alkaline Phosphatase (b-ALP), Osteocalcin and Osteoprotegerin in two groups of patients suffering from osteoarthritis of the Knee or Hip respectively, aiming to correlate these results with the radiographically assessed severity of the disease and the patients’ age. The results between the two groups were also compared. Patients-Methods: Between March 2007 and February 2009, a total of 175 patients suffering from Knee or Hip Osteoarthritis were enrolled in the study. Following proper radiographic evaluation, the osteoarthritic changes of patients were graded by 3 orthopaedic surgeons according to the system of Kellgren and Lawrence; at the same time the serum levels of biochemical markers were determined. Results: Osteoprotegerin was found to be positively correlated with age in both the Knee (r=0.376, p=0.000) and Hip (r=0.425, p=0.001) group, whether Osteocalcin was significantly correlated with the age in the group of Knee Osteoarthritis(r=0.218, p=0.02). No other significant correlation was noted between the serum level of markers and age of patients in both groups. There was not significant difference in the mean serum level of biochemical markers among patients belonging to each of the four different levels of severity of hip and knee OA. There was no significant impact of the type of Osteoarthritis, to the serum level of all biochemical markers. Conclusions: Based on our results, it seems that none of the serum biochemical markers studied can be used (either independently or in combination with the others) as surrogates for radiographic imaging in Hip and Knee osteoarthritis

Background: The clinical significance of bone turnover markers is well recognized, at least in several diseases affecting the bone metabolism. However, their clinical significance (if any) remains still unknown in patients undergoing Total Joint Arthroplasty (TJA). Changes in the levels of some markers have been reported in the early postoperative period after Total Hip Arthroplasty; however their exact postoperative course has not been clearly documented yet. In order to assess the clinical value of biochemical markers when trying to determine the fixation of orthopaedic implants, it is necessary to clarify their normal postoperative course. The aim of this study was to extend the evaluation of the course of bone turnover markers over a longer period (12 postoperative months) following a TJA, and to assess the postoperative course for two of them (RANKL and Osteoprotegerin) for the first time. Methods: The serum levels of RANKL, Osteocalcin, Osteoprotegerin and bALP were determined one day preoperatively and several times during the first postoperative year in patients suffering from idiopathic osteoarthritis that underwent total knee (n=23) and hip arthroplasties (n=24). Results: There were statistically significant changes in the serum levels of all markers over time (p< 0,001). RANKL values initially increased and then gradually decreased. Following an initial decrease, Osteocalcin values continuously increased until the 2nd postoperative month and then continuously decreased. Osteoprotegerin initially increased, then decreased until the 4th postoperative month and then increased again reaching a peak 8 months postoperatively. Bone-specific ALP decreased until the 7th postoperative day. After that time it continuously increased, reaching a peak at the 8th month, and then it gradually decreased. There were no major differences in the postoperative course of all markers between the hip and knee arthroplasties. Conclusions: The levels of all bone markers did not uniformly ‘return’ to their preoperative values one year postoperatively. A one-year period is not enough, when assessing an orthopaedic implant’s fixation with the use of bone turnover markers

Aseptic loosening of orthopaedic implants has a major financial impact on the Health Service. The process is thought to be caused by wear particles that are phagocytosed by macrophages and hence stimulate bone resorption via a cytokine response. Previous work suggests that factors inhibiting or enhancing bone resorption act through regulation of the OPG and RANK-L mechanism. The objective of this study was to identify the role of RANK-L and OPG within the cytokine response leading to orthopaedic implant loosening.

Ten samples of cellular membrane obtained during revision arthroplasty surgery were analysed with basic histological staining, immunohistology and polymerase chain reaction (PCR). In vitro studies were also carried out using explanted cancellous bone, to which PMMA particles were added and bone resorbing osteoclastic cells were identified by their Tartrate-Resistant Acid Phosphatase (TRAP) activity.

PCR identified the presence of OPG in all of the periprosthetic samples, with RANK-L shown in 40% of the specimens. Immunoreactivity was shown for CD3, CD68 and RANK-L. In vitro studies confirm that there is an initial burst of inflammatory cytokine activity that then subsequently plateaus.

A balance of RANK-L and OPG regulates bone resorption at the bone/implant interface of implants by stimulating a significant initial inflammatory response which leads to loosening.

Aims. The present study investigated receptor activator of nuclear factor kappa-Β ligand (RANKL), osteoprotegerin (OPG), and Runt-related transcription factor 2 (RUNX2) gene expressions in giant cell tumour of bone (GCTB) patients in relationship with tumour recurrence. We also aimed to investigate the influence of CpG methylation on the transcriptional levels of RANKL and OPG. Methods. A total of 32 GCTB tissue samples were analyzed, and the expression of RANKL, OPG, and RUNX2 was evaluated by quantitative polymerase chain reaction (qPCR). The methylation status of RANKL and OPG was also evaluated by quantitative methylation-specific polymerase chain reaction (qMSP). Results. We found that RANKL and RUNX2 gene expression was upregulated more in recurrent than in non-recurrent GCTB tissues, while OPG gene expression was downregulated more in recurrent than in non-recurrent GCTB tissues. Additionally, we proved that changes in DNA methylation contribute to upregulating the expression of RANKL and downregulating the expression of OPG, which are critical for bone homeostasis and GCTB development. Conclusion. Our results suggest that the overexpression of RANKL/RUNX2 and the lower expression of OPG are associated with recurrence in GCTB patients. Cite this article: Bone Joint Res 2024;13(2):84–91

Aims. To investigate whether idiopathic osteonecrosis of the femoral head (ONFH) is related to impaired osteoblast activities. Methods. We cultured osteoblasts isolated from trabecular bone explants taken from the femoral head and the intertrochanteric region of patients with idiopathic ONFH, or from the intertrochanteric region of patients with osteoarthritis (OA), and compared their viability, mineralization capacity, and secretion of paracrine factors. Results. Osteoblasts from the intertrochanteric region of patients with ONFH showed lower alkaline phosphatase (ALP) activity and mineralization capacity than osteoblasts from the same skeletal site in age-matched patients with OA, as well as lower messenger RNA (mRNA) levels of genes encoding osteocalcin and bone sialoprotein and higher osteopontin expression. In addition, osteoblasts from patients with ONFH secreted lower osteoprotegerin (OPG) levels than those from patients with OA, resulting in a higher receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) ligand (RANKL)-to-OPG ratio. In patients with ONFH, osteoblasts from the femoral head showed reduced viability and mineralized nodule formation compared with osteoblasts from the intertrochanteric region. Notably, the secretion of the pro-resorptive factors interleukin-6 and prostaglandin E. 2. as well as the RANKL-to-OPG ratio were markedly higher in osteoblast cultures from the femoral head than in those from the intertrochanteric region. Conclusion. Idiopathic ONFH is associated with a reduced mineralization capacity of osteoblasts and increased secretion of pro-resorptive factors. Cite this article: Bone Joint Res 2021;10(9):619–628

Objectives. Enhanced micromotions between the implant and surrounding bone can impair osseointegration, resulting in fibrous encapsulation and aseptic loosening of the implant. Since the effect of micromotions on human bone cells is sparsely investigated, an in vitro system, which allows application of micromotions on bone cells and subsequent investigation of bone cell activity, was developed. Methods. Micromotions ranging from 25 µm to 100 µm were applied as sine or triangle signal with 1 Hz frequency to human osteoblasts seeded on collagen scaffolds. Micromotions were applied for six hours per day over three days. During the micromotions, a static pressure of 527 Pa was exerted on the cells by Ti6Al4V cylinders. Osteoblasts loaded with Ti6Al4V cylinders and unloaded osteoblasts without micromotions served as controls. Subsequently, cell viability, expression of the osteogenic markers collagen type I, alkaline phosphatase, and osteocalcin, as well as gene expression of osteoprotegerin, receptor activator of NF-κB ligand, matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-1, were investigated. Results. Live and dead cell numbers were higher after 25 µm sine and 50 µm triangle micromotions compared with loaded controls. Collagen type I synthesis was downregulated in respective samples. The metabolic activity and osteocalcin expression level were higher in samples treated with 25 µm micromotions compared with the loaded controls. Furthermore, static loading and micromotions decreased the osteoprotegerin/receptor activator of NF-κB ligand ratio. Conclusion. Our system enables investigation of the behaviour of bone cells at the bone-implant interface under shear stress induced by micromotions. We could demonstrate that micromotions applied under static pressure conditions have a significant impact on the activity of osteoblasts seeded on collagen scaffolds. In future studies, higher mechanical stress will be applied and different implant surface structures will be considered. Cite this article: J. Ziebart, S. Fan, C. Schulze, P. W. Kämmerer, R. Bader, A. Jonitz-Heincke. Effects of interfacial micromotions on vitality and differentiation of human osteoblasts. Bone Joint Res 2018;7:187–195. DOI: 10.1302/2046-3758.72.BJR-2017-0228.R1

The effect of high-fat diet and testosterone replacement therapy upon bone remodelling was investigated in orchiectomised male APOE-/- mice. Mice were split in to three groups: sham surgery + placebo treatment (control, n=9), orchiectomy plus placebo treatment (n=8) and orchiectomy plus testosterone treatment (n=10). Treatments were administered via intramuscular injection once a fortnight for 17 weeks before sacrifice at 25 weeks of age. Tibiae were scanned ex-vivo using µCT followed by post-analysis histology and immunohistochemistry. Previously presented µCT data demonstrated orchiectomised, placebo treated mice exhibited significantly reduced trabecular bone volume, number, thickness and BMD compared to control mice despite no significant differences in body weight. Trabecular parameters were rescued back to control levels in orchiectomised mice treated with testosterone. No significant differences were observed in the cortical bone. Assessment of TRAP stained FFPE sections revealed no significant differences in osteoclast or osteoblast number along the endocortical surface. IHC assessment of osteoprotegerin (OPG) expression in osteoblasts is to be quantified alongside markers of osteoclastogenesis including RANK and RANKL. Results support morphological analysis of cortical bone where no change in cortical bone volume or density between groups is in line with no significant change in osteoblast or osteoclast number and percentage across all three groups. Future work will include further IHC assessment of bone remodelling and adiposity, as well as utilisation of mechanical testing to establish the effects of observed morphological differences in bone upon mechanical properties. Additionally, the effects of hormone treatments upon murine-derived bone cells will be investigated to provide mechanistic insights

Aim. Vertebral osteomyelitis (VO) is an infection of the spine mostly caused by bacterial pathogens. The pathogenesis leading to destruction of intervertebral discs (IVD) and adjacent vertebral bodies (VB) is poorly described. We aimed to investigate the connection between infection, bone- and disc-metabolism in VO patients. Method. Fourteen patients with VO (infection group) and 14 patients with incomplete burst fractures of the spine (fracture group as controls) were included prospectively. Demographic data, treatment details, laboratory infection markers, and patient-reported outcome were assessed. Tissue biopsies from affected IVDs and adjacent VBs were analyzed for mRNA-expression levels of 18 target genes including chemokines, adipokines and genes involved in bone-metabolism by RT-qPCR. Results. The Receptor activator of NF-κB/Osteoprotegerin (RANK/OPG) expression ratio was elevated in VB and IVD of the infection group (p<0.001 and p=0.028, respectively). The RANK-ligand (RANKL)/OPG expression ratio was elevated in VB of the infection group (p<0.01). Expressions of the chemokines IL8 and CCL20 were higher in VB samples of the infection group. The expression of leptin was higher in IVD tissue, the mRNA expression of omentin and resistin was lower in VBs of the infection group. OPG mRNA expression was lower in infected VB and in IVD tissue compared to the fracture group. Conclusions. We identified similar expression patterns of pro-inflammatory cytokines and the RANK/RANKL/OPG axis in VBs and IVDs of patients with VO. This finding suggests that common immuno-metabolic pathways are involved in mechanisms leading to tissue degradation in VBs and IVDs during VO

Abstract. Objectives. The mechanisms underlying abnormal joint mechanics are poorly understood despite it being a major risk factor for developing osteoarthritis. This study investigated the response of a 3D in vitro bone cell model to mechanical load. Methods. Human MSC cells (Y201) embedded in 3D type I collagen gels were differentiated in osteogenic media for 7-days in deformable, silicone plates. Gels were loaded once (5000 µstrain, 10Hz, 3000 cycles), RNA extracted 1-hr post load and assessed by RT-qPCR and RNAseq analysis (n=5/treatment). Cell shape and phenotype were assessed by immunocytochemistry and phalloidin staining. Data was analysed by Minitab. Results. RTqPCR revealed cells expressed markers of mature osteocytes (E11, sclerostin, DMP-1) and osteoprotegerin (OPG), alkaline phosphatase and type I collagen (COL1A1). Immunolocalisation of sclerostin and DMP-1 protein along with phalloidin staining confirmed a dendritic osteocyte phenotype. Load almost abolished sclerostin gene expression (p=0.05) and reduced E11 (2-fold p=0.03); COL1A1 was unchanged (p=0.349). Using DEseq2 analysis, of the 981 genes differentially regulated more than 2-fold at FDR p<0.05, 159 were downregulated and 821 upregulated by load. These were involved in processes important in bone biology including the inflammatory response (56 genes), ECM organisation (27), ageing (30), response to mechanical load (23), ER stress (34), regulation of ossification (26), bone morphogenesis (14), cartilage development (14), programmed cell death (161), and positive regulation of bone mineralisation (6). Discussion. Y201 cells were successfully differentiated to osteocytes. The osteocytes’ mechanical response revealed regulation of factors that contribute to bone remodelling and inflammation. Since the biological mechanisms underlying mechanically induced joint degeneration are unclear, there is a need for humanised, cell models to delineate molecular pathways activated by mechanical load. Such pathways may reveal the molecular basis for genetic predispositions to osteoarthritis and identify new therapeutic targets. 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

Extensive osteolysis adjacent to implants is often associated with wear particles of prosthetic material. We have investigated if RANKL, also known as osteoprotegerin ligand, osteoclast differentiation factor or TRANCE, and its natural inhibitor, osteoprotegerin (OPG), may be important in controlling this bone loss. Cells isolated from periprosthetic tissues containing wear particles expressed mRNA encoding for the pro-osteoclastogenic molecules, RANKL, its receptor RANK, monocyte colony-stimulating factor (M-CSF), interleukin (IL)-1β, tumour necrosis factor (TNF)α, IL-6, and soluble IL-6 receptor, as well as OPG. Osteoclasts formed from cells isolated from periprosthetic tissues in the presence and absence of human osteoblastic cells. When osteoclasts formed in the absence of osteoblastic cells, markedly higher levels of RANKL mRNA relative to OPG mRNA were expressed. Particles of prosthetic materials also stimulated human monocytes to express osteoclastogenic molecules in vitro. Our results suggest that ingestion of prosthetic wear particles by macrophages results in expression of osteoclast-differentiating molecules and the stimulation of macrophage differentiation into osteoclasts

Background. Multiple Myeloma is a hematological malignancy of terminally differentiated plasma cells associated with increased osteoclast activity and decreased osteoblast functions. Systemic antiproliferative treatment includes proteasome inhibitors such as bortezomib, a clinical potent antimyeloma agent. Local delivery of biological active molecules via biomaterial composite implants to the site of the lesion has been shown to be beneficial for bone and implant-associated infections. In anticancer treatment local delivery of anticancer agents to the neoplasia via biomaterial carriers has never been reported before. The purpose of the current is to present the concepts and the first in vivo results for proteasome inhibitor composite biomaterials for local delivery of bortezomib to proliferative multiple myeloma bone lesions including concentration measurements at different anatomical regions in a rat model. Methods. 80 female Sprague-Dawley rats were randomised into five different treatment groups (n=16/group): 1) Empty (2) Xerogel-granulat: XG (3) Xerogel-granulat+100mgbortezomib [b]: XG100b (4) Xerogel-granulat+500mgb:XG500b (5) Xerogel-granulat+2500mgb:XG2500b. A 2.5 mm drill hole was then created in the metaphysis of the left femur. The defect was then either filled with the previously mentioned substitutes or left empty to serve as a control. After 4 weeks femora were harvested followed by histological, histomorphometrical and immunohistochemical (BMP2; bone-morphogenic protein 2, OPG; osteoprotegerin, RANKL; Receptor activator of nuclear factor kappa-B ligand, ASMA; alpha smooth muscle actin, ED1;CD68 antibody). TOF-SIMS was used to assess the distribution of released strontium ions. Statistical analysis was done using SPSS software. Data was not found normally distributed and hence Mann-Whitney U with bonferroni correction was used. To avoid type I errors due to unequal variances and group sizes Games-Howell test was also performed

Background. Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. Within the REBORNE project, a multi-center orthopaedic clinical trial was focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union. Methods. Bone-specific alkaline phosphatase (BAP), C-terminal-propeptide type I-procollagen (PICP), osteocalcin (OC), β-Cross-Laps Collagen (CTX), soluble receptor activator of NFkB (RANKL), osteoprotegerin (OPG) were measured by ELISA assays in blood samples of 22 patients at BM collection and at follow-ups (6, 12 and 24 weeks post-surgery). Results. A significant relationship with age was found only at Visit 6, with an inverse correlation for CTX, RANKL and OC, and positive for OPG. BTM levels were not related to gender. As an effect of local regenerative process, some BTM showed significant changes in comparison to the starting value. In particular, the time course of BAP, PICP and RANKL was different in patients with a successful healing in comparison to patients with negative outcome. The BTM profile indicated remarkable bone formation activity after 12 weeks after surgery. However, the paucity of failed patients in our case series did not allow to prove statistically the role of BTM as predictors of the final outcome. Conclusion. BTM related to bone cell function are useful to measure the efficacy of a regenerative approach based on expanded MSC. Level of evidence. Diagnostic Level IV. Work supported by the EC, Seventh Framework Programme (FP7), through the REBORNE Project, grant agreement no. 241879