Bone resorption involves dissolution of mineral and enzymatic degradation of bone matrix. The primary enzyme is cathepsin K but other proteases including matrix metalloproteinases are involved. Some cleavage products of cathepsin K have been partially identified, including crossed-linked telopeptides of type I collagen. However, the pathway of type I bone collagen degradation has not been fully elucidated. The aim of this study was to comprehensively characterise the entire complement of bone breakdown products resulting from osteoclast action under controlled conditions in vitro . Complete characterisation of these breakdown products will advance understanding of osteoclast biology and has the potential to reveal new biomarkers of bone resorption. We analysed extracellular media from osteoclasts cultured on dentine substrates, using untargeted liquid chromatography mass spectrometry. We discovered 22 breakdown products resulting from osteoclastic action. These products were peptide fragment sequences that mapped to various collagen proteins present in bone and dentine matrix. Nine peptide fragments mapped exclusively to collagen I alpha-1 chain (COL1A1), the most abundant protein in bone. Analysis of the reported cleavage sites in the COL1A1 protein sequence indicated 7/9 COL1A1-specific fragments not explained by known proteolytic events. We subsequently showed that 14 of the fragment products were present in human serum and/or urine from metabolomic datasets obtained from patients with the inherited metabolic disease alkaptonuria (serum) and lung cancer (urine). Two products were at higher concentration (P <0.05, fold change >2) in urine from patients with bone metastasis (29/112) from the lung cancer cohort. The range of collagen peptide fragments we discovered as a direct result of osteoclast activity indicates a complexity of bone resorption pathways not previously known. Monitoring the concentrations of these novel bone markers in biofluids has the potential to capture multiple pathways of bone resorption activity beyond the existing assays based on Cathepsin K. Lay summary Breakdown of bone tissue is performed by specialised bone cells called osteoclasts in a process known as bone resorption. Knowledge of specific molecules produced from osteoclasts acting on bone is important for a) understanding bone resorption in health and disease, and b) clinical tests of bone resorption from measurement of these breakdown products. Here we aimed to characterise the entire complement of bone breakdown products resulting from osteoclast action under controlled conditions in the laboratory. We found a total of 22 breakdown products produced from osteoclasts cultured on wafers of dentine, a tissue with almost identical composition to bone. Analysis of the structures of these products revealed fragments of varying size produced from digestion of specific proteins present in bone. We then showed that 14/22 bone protein fragments observed in our cell culture experiments were also present in human blood and/or urine. In our analysis of urine from patients with lung cancer, we found that two of the protein fragments we identified were higher in a subset (29/112) of patients with bone metastasis. Our findings provide new insights into the mechanisms of bone resorption and show the potential for monitoring the range of urine bone protein fragments in bone disorders. ### Competing Interest Statement The authors have declared no competing interest.

JBMR PlusVolume 5, Issue S4 e10499 AbstractsOpen Access Abstracts First published: 14 June 2021 https://doi.org/10.1002/jbm4.10499AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume5, IssueS4Supplement: Bone Research Society BRS 2020 Online 6–8 July 2020June 2021e10499 RelatedInformation

Objective: Alkaptonuria (AKU) is a rare, inherited disorder of tyrosine metabolism, where patients are unable to breakdown homogentisic acid (HGA), which increases systemically over time. It presents with a clinical triad of features; HGA in urine, ochronosis of collagenous tissues, and the subsequent ochronotic arthritis of these tissues. In recent years the advance in the understanding of the disease and the potential treatment of the disorder looks promising with the data on the efficacy of nitisinone. However, there are limited methods for the detection and monitoring of ochronosis in vivo, or for treatment monitoring. The study aim was to test the hypothesis that Raman spectra would identify a distinct chemical fingerprint for the non-ochronotic, compared to ochronotic cartilage. Design: Ochronotic and non-ochronotic cartilage from human hips and ears were analysed using Raman spectroscopy. Results: Non-ochronotic cartilage spectra were similar and reproducible and typical of normal articular cartilage. Conversely, the ochronotic cartilage samples were highly fluorescent and displayed limited or no discernible Raman peaks in the spectra, in stark contrast to their non-ochronotic pairs. Interestingly, a novel peak was observed associated with the polymer of HGA in the ochronotic cartilage that was confirmed by analysis of pigment derived from synthetic HGA. Conclusion: This technique reveals novel data on the chemical differences in ochronotic compared with non-ochronotic cartilage, these differences are detectable by a technique that is already generating in vivo data and demonstrates the first possible procedure to monitor the progression of ochronosis in tissues of patients with AKU. (C) 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

BackgroundAlkaptonuria (AKU) is present from birth, yet clinical effects are considered to appear later in life. Morbidity of AKU, considered irreversible, is secondary to ochronosis. Age of ochronosis onset is not clearly known. Nitisinone profoundly lowers homogentisic acid (HGA), the metabolic defect in AKU. Nitisinone also arrests ochronosis and slows progression of AKU. However, tyrosinaemia post-nitisinone has been associated with corneal keratopathy, rash and cognitive impairment in HT 1. The optimal time to start nitisinone in AKU is unknown.MethodsIn an open, cross-sectional, single-site study, 32 patients with AKU were to be recruited. The primary outcome was presence of ochronosis in an ear biopsy. Secondary outcomes included analysis of photographs of eyes/ears, serum/urine HGA, markers of tissue damage/inflammation/oxidation, MRI imaging, gait, quality of life and Alkaptonuria Severity Score Index (qAKUSSI).ResultsThirty patients, with mean age (SD) 38 (14) years, were recruited. Percentage pigmentation within ear biopsies increased with age. Ear pigmentation was detected in a 20-year-old woman implying ochronosis can start in patients before the age of 20. Gait and qAKUSSI were outside the normal range in all the patients with AKU.ConclusionsOchronosis can be present before age 20 years.

Purpose: Alkaptonuria (AKU) is a rare disorder of tyrosine metabolism in which congenital lack of the enzyme homogentisate 1,2-dioxygenase results in elevated circulating concentrations of homogentisic acid (HGA). In AKU severe, early-onset osteoarthropathy is an inevitable consequence of elevated HGA; HGA is deposited in collagenous tissue throughout the body, particularly cartilage of load-bearing joints. Once deposited, HGA is thought to oxidise and polymerise, resulting in a striking dark pigmentation of cartilage which alters its physicomechanical properties that support normal transmission of load. A notable feature of AKU osteoarthropathy, which is also observed in idiopathic osteoarthritis, is aggressive osteoclast-mediated resorption of the subchondral bone plate and articular calcified cartilage. This study aimed to investigate novel biochemical markers for studying bone resorption in AKU. The markers were established from non-targeted chemical profiling of the extracellular media content from osteoclasts cultured on dentine. Methods: Human osteoclast precursors were cultured on dentine wafers from hippo and walrus tusks in 96-well plates with RANKL (66 ng/ml) and MCSF (33 ng/ml) present (n=6); +RANKL/+dentine. Three control groups were a) osteoclast precursors cultured on plastic with RANKL (+RANKL/-dentine; n=6), b) osteoclast precursors cultured on dentine without RANKL (-RANKL/+dentine; n=6) and c) osteoclast precursors cultured on plastic without RANKL (-RANKL/-dentine; n=3). After 14 days culture medium was sampled and analysed on an Agilent 1290 UHPLC coupled to an Agilent 6550 quadrupole time-of-flight mass spectrometer (QTOF-MS). Reversed phase HPLC was performed on a Waters Atlantis dC18 3 μm column with linear gradient elution; 5-95% methanol over 1-12 mins, flow rate 0.4 mL/min. Mass spectrometric analysis was performed in positive polarity electrospray ionisation, mass range 50-1700. Chemical signals were extracted from the data by non-targeted recursive feature extraction using Mass Hunter Profinder (B.08; Agilent). Statistical analyses were performed with Mass Profiler Professional (B.14.00; Agilent). Results: Profiles obtained from media were compared between the +RANKL/+dentine condition and the three control groups. 147 positive ion chemical entities were detected across all culture groups. 59 entities showed statistically significant differences in profiles (p <0.05, fold change >2) across the four culture groups. The clearest difference was in the profiles of media from +RANKL/+dentine compared to the other conditions (Figure 1). 28 unique entities (mass range = 144-1541 Da) were increased (fold change >8) in +RANKL/+dentine media compared with all control groups, and therefore indicative of bone resorption. Based on high-resolution accurate mass, putative identifications for these markers were the collagen tri-peptide glycylprolylhydroxyproline and di-peptides prolyl-glycine, valyl-glycine and tyrosyl-proline. Subsequently, glycylprolylhydroxyproline, prolyl-glycine, valyl-glycine and tyrosyl-proline were detected in profiling data from AKU serum (human) and urine (human and mouse) analysed under identical LC-QTOF-MS conditions; matched accurate mass (±10 ppm) and retention time (±0.3 mins). Conclusions: Non-targeted extracellular chemical profiling of media from osteoclasts cultured on dentine identified clear, novel and specific in vitro markers of bone resorption. High-resolution accurate mass indicated that some of these markers were tri- and di- peptides of collagen. Four of the peptide-containing compounds were identified in AKU urine and serum, supporting them as markers to study and monitor bone resorption associated with osteoarthropathy in AKU.

Purpose: Alkaptonuria is a rare genetically inherited form of OA which is characterised by urine which darkens on standing, pigmentation of articular cartilages and early-onset, rapidly progressing osteoarthropathy of multiple weight bearing joints. The pigmentation is caused by presence of a tyrosine metabolite; homogentisic acid (HGA), which polymerises over time to for a pigment in the articular cartilages. The presence of pigment changes the biochemical and biomechanical properties of the cartilage causing anatomical and physiological dysfunction. To date there is no evidence to suggest the exact time when ochronosis and therefore pathological change in tissues commences. Our study will examine ochronotic and non-ochronotic cartilage to determine if Raman spectroscopy can detect pigmentation in macroscopically non-ochronotic cartilage. It will also compare the spectra of ochronotic and non-ochronotic cartilage to normal cartilage spectra in the current literature. Methods: Cartilage samples were obtained as surgical waste with informed patient consent following ethical approval. The samples were dissected immediately following surgery into ochronotic and non-ochronotic pairs and stored unfixed at -80 °C. Individual cartilage samples were placed on to calcium fluoride discs for Raman spectral analysis. Spectra were acquired using an InVia Raman microspectrometer (Renishaw plc, UK) with an 785 nm laser. Spectra were collected using 20 s and 3 accumulations, at 100 % power (∼10mW at sample) from the non-ochronotic tissue. However, applying the same settings to the ochronotic tissue resulted in detector saturation. Therefore spectra were collected using 1 s and 10 accumulations, at 100 % power. Data were baseline corrected, using polynomial subtraction, to remove fluorescence and normalised. Results: Macroscopically non-ochronotic cartilage displayed spectra typical of normal articular cartilage with peaks corresponding to all major functional groups and amino acids: proline, hydroxyproline, phenylalanine, amide I (carbonyl group) and amide III (C-N and N-H). Cartilage which showed observable macroscopic ochronotic pigmentation and those which were completely ochronotic displayed an absence of a recognisable spectra with no discernible peaks corresponding to functional groups seen in the non-ochronotic sample, but were highly fluorescent. The macroscopically pigmented sample showed two peaks, previously described in the literature, corresponding to aromatic C-C twisting and phosphatidylinositol (lipid). Conclusions: Our data demonstrates that the semi-pigmented and pigmented samples of cartilage were in stark contrast to the non-pigmented samples. While it was relatively straightforward to collect spectra from the non-ochronotic cartilage, which produced ‘typical’ cartilage spectra, the ochronotic cartilage was very fluorescent, which masked the Raman signal. Therefore, there were very few identifiable peaks; two were identified at 626 and 775 cm-1, but were not present in the non-pigmented spectra. The presence of these peaks may be due to the HGA polymer or an alteration to the cartilage. The lack of prominent peaks in the samples is most likely due to a high level of fluorescence that appears to occur when the HGA polymer becomes incorporated within the cartilage matrix. As a result it was not possible to discern how specifically the chemical structure had changed, just that there had indeed been an alteration in structure. This data shows the significant biochemical and structural changes that occur focally in AKU cartilage and contribute to its rapid and devastating progression. Our results suggest Raman spectroscopy may have application in the development of a non-invasive test for AKU severity.

Alkaptonuria is a rare genetic disorder characterized by a high level of circulating (and urine) homogentisic acid (HGA), which contributes to ochronosis when it is deposited in connective tissue as a pigmented polymer. In an observational study carried out by National AKU Centre (NAC) in Liverpool, a total of thirty-nine AKU patients attended yearly visits in varying numbers. At each visit a mixture of clinical, joint and spinal assessments were carried out and the results calculated to yield an AKUSSI (Alkaptonuria Severity Score Index), see “Nitisinone arrests ochronosis and decreases rate of progression of Alkaptonuria: evaluation of the effect of nitisinone in the United Kingdom National Alkaptonuria Centre” (Ranganath at el., 2018). The aim of this data article is to produce visual representation of the change in the components of AKUSSI over 3 years, through radar charts. The metabolic effect of nitisinone is shown through box plots.

Question: Does Nitisinone prevent the clinical progression of the Alkaptonuria? Findings: In this observational study on 39 patients, 2 mg of daily nitisinone inhibited ochronosis and significantly slowed the progression of AKU over a three-year period. Meaning: Nitisinone is a beneficial therapy in Alkaptonuria. Background: Nitisinone decreases homogentisic acid (HGA), but has not been shown to modify progression of Alkaptonuria (AKU). Methods: Thirty-nine AKU patients attended the National AKU Centre (NAC) in Liverpool for assessments and treatment. Nitisinone was commenced at Vi or baseline. Thirty nine, 34 and 22 AKU patients completed 1, 2 and 3 years of monitoring respectively (V2, V3 and V4) in the VAR group. Seventeen patients also attended a pre baseline visit (VO) in the VAR group. Within the 39 patients, a subgroup of the same ten patients attended VO, Vi, V2, V3 and V4 visits constituting the SAME Group. Severity of AKU was assessed by calculation of the AKU Severity Score Index (AKUSSI) allowing comparison between the pre-nitisinone and the nitisinone treatment phases. Results: The ALL (sum of clinical, joint and spine AKUSSI features) AKUSSI rate of change of scores/patient/ month, in the SAME group, was significantly lower at two (0.32 0.19) and three (0.15 0.13) years postnitisinone when compared to pre-nitisinone (0.65 0.15) (p <.01 for both comparisons). Similarly, the ALL AKUSSI rate of change of scores/patient/month, in the VAR group, was significantly lower at one (0.16 0.08) and three (0.19 0.06) years post-nitisinone when compared to pre-nitisinone (0.59 0.13) (p <.01 for both comparisons). Combined ear and ocular ochronosis rate of change of scores/patient/month was significantly lower at one, two and three year's post-nitisinone in both VAR and SAME groups compared with prenitisinone (p <.05). Conclusion: This is the first indication that a 2 mg dose of nitisinone slows down the clinical progression of AKU. Combined ocular and ear ochronosis progression was arrested by nitisinone.

High density mineralised protrusions (HDMP) from the tidemark mineralising front into hyaline articular cartilage (HAC) were first described in Thoroughbred racehorse fetlock joints and later in Icelandic horse hock joints. We now report them in human material. Whole femoral heads removed at operation for joint replacement or from dissection room cadavers were imaged using magnetic resonance imaging (MRI) dual echo steady state at 0.23 mm resolution, then 26‐μm resolution high contrast X‐ray microtomography, sectioned and embedded in polymethylmethacrylate, blocks cut and polished and re‐imaged with 6‐μm resolution X‐ray microtomography. Tissue mineralisation density was imaged using backscattered electron SEM (BSE SEM) at 20 kV with uncoated samples. HAC histology was studied by BSE SEM after staining block faces with ammonium triiodide solution. HDMP arise via the extrusion of an unknown mineralisable matrix into clefts in HAC, a process of acellular dystrophic calcification. Their formation may be an extension of a crack self‐healing mechanism found in bone and articular calcified cartilage. Mineral concentration exceeds that of articular calcified cartilage and is not uniform. It is probable that they have not been reported previously because they are removed by decalcification with standard protocols. Mineral phase morphology frequently shows the agglomeration of many fine particles into larger concretions. HDMP are surrounded by HAC, are brittle, and show fault lines within them. Dense fragments found within damaged HAC could make a significant contribution to joint destruction. At least larger HDMP can be detected with the best MRI imaging ex vivo.

Purpose: There is convincing evidence that extracellular ATP, signalling through P2 purinoceptors, plays a major role in the regulation of bone remodelling particularly in mechanotransduction. P2 receptors are known to be expressed in cartilage but their role in regulating chondrocyte physiology is still largely unexplored. The aim of this research was to identify which P2 receptors are expressed in chondrocytes, and to determine the consequences of receptor activation. Methods: Initial experiments were undertaken on the C20 chondrocyte cell line, cultured in monolayer or alginate beads. Subsequently, we investigated primary human chondrocytes isolated by enzyme digestion. Primary chondrocytes were cultured in 1, 5 or 20% oxygen. P2 receptor expression was determined by RT-PCR. ATP concentration in conditioned medium was measured using the luciferin/luciferase assay in a Berthold Tube Luminometer. Lactate was measured using a colorimetric kit supplied by Cobas. Results: Chondrocytes expressed a range of P2 receptors including P2Y1, P2Y2, P2Y4, P2Y6 and P2X7. Addition of ATP had little effect on the production of extracellular matrix by chondrocytes. However, there were biphasic and time dependent effects on cell proliferation. Addition of ATP at concentration below 1 micromolar led to an initial increase in cell number, whereas addition of 10-100 micromolar ATP resulted in a dose-dependent decrease in cell number. One of the most striking effects of ATP treatment was a dose dependent acidification of the culture medium over the first 24 hours following initial exposure. Further investigation revealed that the acidification was the result of an increase in lactate production. Treatment with 100 micromolar ATP induced a massive release of ATP from the cells, which would have effectively depleted them of ATP. The release of ATP appeared to be a result of P2X7 activation as it could be replicated by addition of 10 micromolar BzATP, a prototypic P2X7 receptor agonist with 10 fold greater potency than ATP. The ATP-induced ATP release was observed in both C20 and primary chondrocytes, and was unaffectedby oxygen tension. Conclusions: The results of this study demonstrate that chondrocytes express a range of P2 receptors including P2X7. This latter is a pore- and channel-forming receptor which can induce proliferation or apoptosis. Low concentrations of extracellular ATP appear to have a positive effect on chondrocyte cell number, indicating that there might be a trophic effect on cell growth/survival. However when extracellular ATP was elevated to concentrations which might occur following localised mechanical injury or inflammation, activation of P2X7 receptors resulted in release of more ATP. Diffusion of released ATP to adjacent chondrocytes could lead to further activation of P2X7 receptor possibly resulting in the death of cells close to lesions. Diffusion of ATP through cartilage is likely to play a significant role in regulating cell function in this aneural, avascular tissue

Icariside II is considered one of the most important natural flavonoids with multiple bioactivities from traditional Chinese medicine Yin Yanghuo (YYH) or Horny Goat Weed (Epimedium koreanum Nakai). Previous studies show that Icariside II exhibits potent cytotoxicity against a broad spectrum of human cancer cells through various signaling transduction pathways. However, there are few reports about the effect of Icariside II on osteosarcoma cell. In this study, we found that Icariside II decreased cell proliferation in human osteosarcoma MG-63 cells and human osteosarcoma Saos-2 cells. In addition, Icariside II inactivated EGFR/mTOR signaling pathway, including EGFR, PI3K/AKT/PRAS40, Raf/MEK/ERK as well as mTOR. Furthermore, Icariside II inhibited epidermal growth factor (EGF)-induced activation of EGFR/mTOR signaling pathway. Pretreatment of EGF partially reversed cell viability decreased by Icariside II. Importantly, Icariside II inhibited the proliferation of transplantable tumors and EGFR/mTOR signaling pathway in sarcoma-180 bearing mice. In summary, these results indicate that Icariside II inhibits the proliferation of osteosarcoma cells in vitro and in vivo via EGFR/mTOR signaling pathway.