Objectives. The objectives of this study were: 1) to examine osteophyte formation, subchondral bone advance, and bone marrow lesions (BMLs) in osteoarthritis (OA)-prone Hartley guinea pigs; and 2) to assess the disease-modifying activity of an orally administered phosphocitrate ‘analogue’, Carolinas Molecule-01 (CM-01). Methods. Young Hartley guinea pigs were divided into two groups. The first group (n = 12) had drinking water and the second group (n = 9) had drinking water containing CM-01. Three guinea pigs in each group were euthanized at age six, 12, and 18 months, respectively. Three guinea pigs in the first group were euthanized aged three months as baseline control. Radiological, histological, and immunochemical examinations were performed to assess cartilage degeneration, osteophyte formation, subchondral bone advance, BMLs, and the levels of matrix metalloproteinse-13 (MMP13) protein expression in the knee joints of hind limbs. Results. In addition to cartilage degeneration, osteophytes, subchondral bone advance, and BMLs increased with age. Subchondral bone advance was observed as early as six months, whereas BMLs and osteophytes were both observed mainly at 12 and 18 months. Fibrotic BMLs were found mostly underneath the degenerated cartilage on the medial side. In contrast, necrotic BMLs were found almost exclusively in the interspinous region. Orally administered CM-01 decreased all of these pathological changes and reduced the levels of MMP13 expression. Conclusion. Subchondral bone may play a role in cartilage degeneration. Subchondral bone changes are early events; formation of osteophytes and BMLs are later events in the OA disease process. Carolinas Molecule-01 is a promising small molecule candidate to be tested as an oral disease-modifying drug for human OA therapy. Cite this article: Y. Sun, A. J. Kiraly, A. R. Sun, M. Cox, D. R. Mauerhan, E. N. Hanley Jr. Effects of a phosphocitrate analogue on osteophyte, subchondral bone advance, and bone marrow lesions in Hartley guinea pigs. Bone Joint Res 2018;7:157–165. DOI:10.1302/2046-3758.72.BJR-2017-0253

Despite the growing success of OCA transplantation in treating large articular cartilage lesions in multiple joints, revisions and failures still occur. While preimplantation subchondral drilling is intended to directly decrease allograft bioburden and has been associated with significant improvements in outcomes after OCA transplantation, the effects of size, number, and spacing of subchondral bone drill sites have not been fully evaluated. This study aimed to investigate the effects of drill size with or without pulse-lavage of OCA subchondral bone by quantifying remnant marrow elements using histomorphometry. With IRB and ACUC approvals, human and canine OCAs were acquired for research purposes. Portions of human tibial plateau OCAs acquired from AATB-certified tissue banks that would otherwise be discarded were recovered and sectioned into lateral and medial hemiplateaus (n=2 each) with a thickness of 7 mm. Canine femoral condyles and tibial plateaus were split into lateral and medial components with a thickness of 7 mm (n=8). Using our clinical preimplantation preparation protocol, holes were drilled into the subchondral bone of each condyle and hemiplateau OCA using either 1.6 mm OD or 3.2 mm OD drill bits from the cut surface to the cortical subchondral bone plate. One femoral condyle and one hemiplateau per drill bit size were pulse-lavaged while the corresponding OCAs were not. The mean total %-fill remaining marrow elements for each treatment group was calculated. Little to no quantifiable bone marrow element retention was noted to remain within the subchondral bone of human or canine OCA specimens after subchondral drilling of allograft bone with either drill bit size evaluated and with or without pulse-lavage. The %-fill was consistent across zones, ranging from 1-5%. This project was designed to provide a preliminary histologic evaluation of the effects of drill size on OCA preimplantation preparation efficacy based on amount of remaining bone marrow elements in human and canine femoral condyle and tibial plateau specimens. Based on these initial findings, choice of drill bit size for OCA subchondral drilling may need to be based on the associated biomechanical effects rather than effects on donor bone marrow element removal

Cartilage diseases have a significant impact on the patient's quality of life and are a heavy burden for the healthcare system. Better understanding, early detection and proper follow-up could improve quality of life and reduce healthcare related costs. Therefore, the aim of this study was to evaluate if difference between osteoarthritic (OA) and non-osteoarthritic (non-OA) knees can be detected quantitatively on cartilage and subchondral bone levels with advanced but clinical available imaging techniques. Two OA (mean age = 88.3 years) and three non-OA (mean age = 51.0 years) human cadaveric knees were scanned two times. A high-resolution peripheral quantitative computed tomography (HR-pQCT) scan (XtremeCT, Scanco Medical AG, Switzerland) was performed to quantify the bone microstructure. A contrast-enhanced clinical CT scan (GE Revolution Evo, GE Medical Systems AG, Switzerland) was acquired with the contrast agent Visipaque 320 (60 ml) to measure cartilage. Subregions dividing the condyle in four parts were identified semi-automatically and the images were segmented using adaptive thresholding. Microstructural parameters of subchondral bone and cartilage thickness were quantified. The overall cartilage thickness was reduced by 0.27 mm between the OA and non-OA knees and the subchondral bone quality decreased accordingly (reduction of 33.52 % in BV/TV in the layer from 3 to 8 mm below the cartilage) for the femoral medial condyle. The largest differences were observed at the medial part of the femoral medial condyle both for cartilage and for bone parameters, corresponding to clinical observations. Subchondral bone microstructural parameters and cartilage thickness were quantified using in vivo available imaging and apparent differences between the OA and non-OA knees were detected. Those results may improve OA follow-up and diagnosis and could lead to a better understanding of OA. However, further in vivo studies are needed to validate these methods in clinical practice

µCT images are commonly analysed to assess changes in bone density and architecture in preclinical murine models. Several platforms provide automated analysis of bone architecture parameters from volumetric regions of interest (ROI). However, segmentation of the regions of subchondral bone to create the volumetric ROIs remains a manual and time-consuming task. This study aimed to develop and evaluate automated pipelines for trabecular bone architecture analysis of mouse proximal tibia subchondral bone. A segmented dataset involving 62 knees (healthy and arthritic) from 10-week male C57BL/6 mice were used to train a U-Net type architecture, with µCT scans (downsampled) input that output segmentation and bone volume density (BV/TV) of the subchondral trabecular bone. Segmentations were upsampled and used in tandem with the original scans (10µ) as input for architecture analysis along with the thresholded trabecular bone. The analysis considered the manually and U-Net segmented ROIs using two available pipelines: the ITKBoneMorphometry library and CTan (SKYSCAN). The analyses included: bone volume (BV), total volume (TV), BV/TV, trabecular number (TbN), trabecular thickness (TbTh), trabecular separation (TbSp), and bone surface density (BSBV). There was good agreement for bone measures between the manual and U-Net pipelines utilizing ITK (R=0.88-0.98) and CTan (R=0.91-0.98). ITK and CTan showed good agreement for BV, TV, BV/TV, TbTh and BSBV (R=0.9-0.98). However, a limited agreement was seen between TbN (R=0.73) and TbSb (R=0.59) due to methodological differences in how spacing is evaluated. This U-Net/ITK pipeline seamlessly automated both segmentation and quantification of the proximal tibia subchondral bone. This automated pipeline allows the analysis of large volumes of data, and its open-source nature may enable the standardization of stereologic analysis of trabecular bone across different research groups

Abstract. Objectives. Osteoarthritis (OA) of the knee causes pain, limits activity and impairs quality of life. Raman microspectroscopy can provide information about the chemical changes that occur in OA, to enhance our understanding of its pathology. The objective of this study is to detect OA severity in human cartilage and subchondral bone using Raman microspectroscopy and explore corresponding mechanical properties of the subchondral bone. Methods. OA tibial plateaus were obtained from total knee replacement surgery with REC (18/LO/1129) and HRA approval. Medial tibial plateau, representing a major weight-bearing area, was graded according to the International Cartilage Repair Society (ICRS) scoring system. Nine samples (3 samples of each graded as moderate, severe and very severe) were selected for Raman and mechanical analyses. Results. A decrease in Raman intensity of glycosaminoglycan (GAG) CH3 (1380cm-1), collagen amide I (1655cm-1) and CH2 deformation (1450cm-1) was observed in cartilage with increasing severity. The calcified cartilage showed a prominent mineral peak at 959cm-1 in the Raman spectra. Meanwhile, an increase of the Raman intensity of collagen amide I (1655 cm-1) and CH2 deformation (1450cm-1), full width half maximum (FWHM) of the mineral peak (960cm-1) and elastic modulus was observed in subchondral bone with increasing severity. Carbonate-to-phosphate ratio (960/1070cm-1) decreased with disease severity. Conclusions. In conclusion, as OA severity increases, cartilage loses GAG and collagen matrix, while bone increases its collagen matrix, with reduction in mineral crystallinity that cause increase of the elastic modulus. Detection of matrix and mineral changes by Raman microspectroscopy would facilitate the identification of OA severity, and potentially progression, and pave the way towards developing treatment. 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

Osteoarthritis (OA) is the most common musculoskeletal disease in the EU and is characterized by cartilage degeneration, pain and restricted movement. Post-Traumatic OA (PTOA) is a specific disease subset that occurs subsequent to traumatic injury, such as ACL rupture and makes up 12% of the overall disease burden. Our current understanding PTOA is that initial injury affects multiple tissues, and many/all contribute to overall ‘joint failure.’ MRI scans show that subchondral bone marrow lesions (BMLs) are present in 80% of ACL rupture cases in the immediate aftermath of joint injury. Their presence indicates an acute consequence in subchondral bone. It has also been suggested that BMLs overlap with, or directly represent, bone microdamage. Microdamage is known to induce osteoclast-mediated remodelling in bone. Therefore, the inhibition of subchondral bone remodelling, particularly in the early phase post-injury, may be a candidate therapeutic approach for preventing PTOA. Finally, the contiguous link between subchondral bone and articular cartilage, can allow transport of small molecules across this boundary, this suggests that bone/cartilage crosstalk is likely to be a key factor in PTOA development after injury. This presentation will summarize recent advances in our understanding these phenomena in both animal and human studies

Osteoarthritis (OA) affects bone cartilage and underlying bone. Mechanically, the underlying bone provides support to the healthy growth of the overlying cartilage. However, with the progress of OA, bone losses and cysts occur in the bone and these would alter the biomechanical behaviour of the joint, and further leading to bone remodelling adversely affect the overlying cartilage. Human femoral head and femoral condyle were collected during hip or knee replacement operation due to the end stage of osteoarthritis (age 50–70), and the cartilage patches were graded and marked. A volunteer patient, with minor cartilage injury in his left knee while the right knee is intact, was used as control. Peripheral quantitative computed tomography (pQCT) was used to scan the bone and to determine the volumetric bone mineral density (vBMD) distribution. The examination of retrieved tissue explants from osteoarthritic patients revealed that patches of cartilage were worn away from the articular surface, and patches of intact cartilage were left. The cysts, ranging from 1 to 10mm were existed in all osteoarthritic bones, and were located close to cartilage defects in the weight-bearing regions, and closely associated with the grade of cartilage defect as measured by pQCT. The bone mineral density (vBMD) distribution demonstrated that the bones around cysts had much higher vBMD than the trabecular bone away from the cysts. Compared to the subchondral bone under thicker cartilage, subchondral bone within cartilage defect has higher vBMD. This may result from the mechanical stimulation as a result of bone-bone direct contact with less protection of cartilage in cartilage defect regions. This study showed an association between cartilage defect and subchondral bone mineral density distribution. Cysts were observed in all osteoarthritic samples and they are located close to cartilage defects in the weight-bearing regions. Cartilage defect altered the loading pattern of the joints, this leading to the bone remodelling and resultant bone structural changes as compared to the normal bone tissues. This work was financially supported by The ARUK Proof of Concept Award (grant no: 21160)

Osteoarthritis is associated with changes to the matrix composition of subchondral bone. Raman spectroscopy has the potential to detect in vivo the molecular changes in osteoarthritic subchondral bone. The objectives were to determine the levels of mineralisation, carbonate accumulation and bone remodelling in osteoarthritic subchondral bone, which we defined as within 3mm of articular cartilage. This was compared to the proximal-compartment (10mm distal to articular cartilage) and the head-neck junction. Five osteoarthritic (average age: 76 years) and five normal cadaveric femoral heads (average age: 72 years) were scanned using peripheral quantitative computed tomography and then sectioned coronally. Raman spectroscopy was then used to scan the femoral heads. All scans were done in the plane of the longitudinal axis of the diaphysis. Cores were subsequently extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis performed to determine the levels of homotrimeric collagen. The phosphate-to-amide I ratio, from the Raman spectra, in osteoarthritic subchondral bone was significantly greater than controls (p=0.023). Within osteoarthritic specimens, the phosphate-to-amide I ratio increased proximally. The density in osteoarthritic subchondral bone was 89mg/cm3 higher than controls (p=0.022), and 494mg/cm3 higher than the osteoarthritic proximal-compartment (p<0.001). Moreover, carbonate substitution into the apatite crystals decreased in osteoarthritic specimens. The carbonate-to-amide I ratio was highest in osteoarthritic subchondral bone. Furthermore, the median α1-to-α2-chain ratio in osteoarthritic specimens was 2:1. The changes found in subchondral bone are important in the pathogenesis of osteoarthritis. This study shows that Raman spectroscopy can detect differences between osteoarthritic specimens and controls, further supporting its potential use in diagnosing bone disorders

Summary Statement. Cross-talk between cells from immune and bone system might play a role in molecular regulation of subchondral bone sclerosis in osteoarthritis. Macrophages, B-lymphocytes and tartrate-resistant acid phosphatase activity are specifically increased in sclerotic subchondral bone of patients with knee osteoarthritis. Background. Recent investigations have provided substantial evidence that distinct molecular and morphological changes in subchondral bone tissue, most notably sclerosis, play an active and important role in the pathogenesis of OA. The cellular and molecular regulation of this pathological process remains poorly understood. Here, we investigated whether osteoimmunology, the reciprocal signaling between cells from the immune and bone system, is involved in OA subchondral bone sclerosis. Patients & Methods. Tibial plateaus and informed consent were obtained from patients undergoing total knee arthroplasty due to end-stage OA. Subchondral bone mineralization distribution was analyzed using computed tomography osteoabsoptiometry (CT-OAM) and standardised cryosections of low (non-sclerotic) and high (sclerotic) bone mineralization were prepared (n=18 each). Cartilage degeneration was graded in Safranin-O-stained sections using the Mankin scoring system. The presence of T-lymphocytes, B-cells and macrophages was assessed using immunohistochemical staining of their respective surface markers CD3, CD20 and CD68. Osteoclast activity was visualised by staining of the enzyme marker tartrate-resistant acid phosphatase (TRAP). Cellular characterization of ex vivo subchondral bone outgrowth cultures was performed using alkaline phosphatase (ALP), TRAP staining. Correlation between histological parameters was assessed using Spearman's rank correlation. Statistical differences were calculated using Wilcoxon signed rank test or paired t-test, where appropriate. Results. CT-OAM revealed a heterogeneous distribution of subchondral bone mineralization in OA tibial plateaus, displaying focal areas of sclerosis that overlapped macroscopically with areas of cartilage damage. These data were confirmed at the histological level by a strong correlation between Mankin score and grade of sclerosis (r=0.7, p<0.001). Immunohistochemistry showed that CD20. +. , but not CD3. +. , lymphocytes and CD68. +. mononuclear (macrophage) and multinucleated (osteoclast) cells were present in subchondral marrow spaces. Notably, the number of CD20. +. lymphocytes and CD68. +. cells was significantly (p<0.05) increased in sclerotic subchondral bone. Enhanced osteoclast activity was confirmed by a significantly increased (p<0.05) number of multinucleated and mononuclear TRAP. +. cells in sclerotic bone. Finally, the number of CD68. +. cells was strongly correlated (p<0.001) with Mankin score (r=0.7), grade of sclerosis (r=0.8), CD20. +. lymphocytes (r=0.8), and TRAP-positive cells (r=0.9). Outgrowth cultures of subchondral bone showed cells of different morphologies including fibroblast-shaped osteoblasts and macrophage-like cells. Expression of ALP was detected in the prior, while TRAP expression was evident in the latter. Corresponding with histological analyses, the number of TRAP. +. cells was increased in ex vivo outgrowth cultures of sclerotic compared to non-sclerotic subchondral bone. Conclusions. Together, our data suggest that osteoimmunological mechanisms, specifically the interaction of CD68. +. macrophages with bone-resident cells, play a - previously unknown - role in regulating subchondral bone sclerosis in progressive OA. Targeting osteoimmunology might hold potential as a disease-modifying treatment for OA

Abstract. Objectives. In the human knee, the cells of the articular cartilage (AC) and subchondral bone (SB) communicate via the secretion of biochemical factors. Chondrocyte-based AC repair strategies, such as articular chondrocyte implantation, are widely used but there has been little investigation into the communication between the native SB cells and the transplanted chondrocytes. We hypothesise that this communication depends on the health state of the SB and could influence the composition and quality of the repair cartilage. Methods. An indirect co-culture model was developed using transwell inserts, representing a chondrocyte/scaffold-construct for repair of AC defects adjoining SB with varying degrees of degeneration. Donor-matched populations of human bone-marrow derived mesenchymal stromal cells (BM-MSCs) were isolated from the macroscopically and histologically best and worst osteochondral tissue, representing “healthy” and “unhealthy” SB. The BM-MSCs were co-cultured with normal chondrocytes suspended in agarose, with the two cell types separated by a porous membrane. After 0, 7, 14 and 21 days, chondrocyte-agarose scaffolds were assessed by gene expression and biochemical analyses. Results. Matched healthy and unhealthy BM-MSCs from five patients undergoing knee arthroplasty (2 male, 3 female; 72.8±2.2. SD. years-old) were used, together with normal chondrocytes from a healthy patient (male; 24 years-old). At day 21, there was significantly more glycosaminoglycan per chondrocyte in the scaffolds co-cultured with healthy BM-MSCs (4.37×10. −4. μg/cell±2.69×10. −5. SEM. ) than in those cultured with unhealthy BM-MSCs (3.52×10. −4. μg/cell±2.19×10. −5. SEM. ; p<0.001). Co-culture with unhealthy BM-MSCs caused a difference in expression of COL2A1 (day 0–21 fold change; unhealthy:-32.8±12.9. SEM. ; healthy:-7.82±4.46. SEM. ; p<0.001) and ACAN (unhealthy:+1.51±0.51. SEM. ; healthy:+4.05±0.49. SEM. ; p=0.002). Conclusions. Co-culture with unhealthy BM-MSCs caused a reduction in GAG deposition and expression of genes encoding matrix-specific proteins, compared to culturing with healthy BM-MSCs. There are clinical implications for cell-based cartilage repair, where the health of the SB may influence the outcome of chondrocyte-based therapies

Glutamate is a neurotransmitter that transmits mechanical signals in bone (1) and activates glutamate receptors and transporters, in bone, cartilage, meniscus and synovium (2). Glutamate receptor activation influences inflammatory, degenerative and nociceptive pathways in arthritic joints (2). Thus glutamate signalling is a mechanism whereby mechanical load can directly influence joint pathology and pain. We have investigated components of glutamate signalling in the subchondral bone of patients with osteoarthritis to determine which are expressed and whether this varies in anatomical regions subject to different loads. Subchondral bone was sampled from tibial cuts derived from total knee arthroplasty (n=2, TKR, Kellgren Lawrence grade 3) and from tibial drill hole sites from high tibial osteotomy (n=5, HTO, KL grades 2 and 3) for osteoarthritis. RNA was extracted, reverse transcribed and RT-PCR performed for a housekeeping gene GAPDH, a glutamate transporters (EAAT-1, EAAT1ex9skip), glutamate receptors (NR2A and KA1), a bone matrix protein, osteocalcin, and signaling molecules (osteoprotegerin [OPG], RANKL). We found differential mRNA expression in different regions of subchondral bone. In one TKR patient, EAAT-1 expression was significantly reduced in the anterior zone versus the middle or posterior zones of the tibial plateau (ANOVA, p<0.001). HTO bone cores were subdivided medial/lateral and anterior/posterior. Good quality RNA was obtained from bone cores removed from drill holes during HTO surgery, with GAPDH, osteocalcin, EAAT-1, EAAT1ex9skip, NR2A, KA1, OPG and RANKL mRNA expression detected. In one patient, comparison of gene expression in bone cores obtained pre and post HTO revealed that EAAT1ex9skip was rarely detected in post-op bone whereas KA1 was rare in pre-op bone. This differential mRNA expression may be due to the altered loading through the joint caused by the osteotomy, although these on/off differences need to be quantified to confirm this. We have shown that glutamate transporters and receptors are expressed in human subchondral bone. Activation of these receptors and transporters by the increased synovial fluid concentrations of glutamate released in arthritis will influence pathological changes and nociception. In some patients, glutamate transporter mRNA expression appears to vary with anatomical location in bone, or after HTO surgery, consistent with our original discovery of this transporter as mechanically-regulated in bone (1). If glutamatergic signaling is mechanically regulated in the human knee, this will vary during arthritic disease progression and after joint realignment, providing a direct mechanism linking mechanical loading through the joint to pathology and pain in arthritis

Little is known on how sensory nerves and osteoclasts affect degenerative processes in subchondral bone in osteoarthritis (OA). Substance P (SP) effects on bone are ambivalent but physiological levels are critical for proper bone quality whereas α-calcitonin gene-related peptide (αCGRP) has anabolic effects. Here, we aimed to analyse the influence of an altered sensory neuropeptide microenvironment on subchondral bone in murine OA. Transection of the medial meniscotibial ligament (DMM) of the right hind leg induced joint instability leading to development of OA. Subchondral bone of tibiae from wildtype (WT), alendronate-treated WT (ALN, osteoclast inhibition), αCGRP- and SP- (Tachykinin (Tac)1) knockout mice was analysed by micro-computed tomography 4 and 12 weeks after DMM or sham surgery. Bone resorption marker CTX-I was measured in serum. We observed osteophytosis in all DMM groups and ALN sham mice 4 weeks after surgery but also in sham groups 12 weeks after surgery. In subchondral bone, bone volume density (BV/TV) increased from 4 to 12 weeks after surgery in DMM WT and Tac1-/− mice. DMM WT mice additionally had increased trabecular numbers (Tb.N.) and decreased trabecular space (Tb.Sp.) over time. Sham mice also showed time-dependent alterations in subchondral bone. In sham WT and αCGRP-/− mice specific bone surface (BS/BV) decreased and trabecular thickness (Tb.Th.) increased from 4 to 12 weeks after surgery while subchondral BV/TV of αCGRP-/− mice increased. Comparison of subchondral bone parameters at each time point showed elevated BV/TV in ALN DMM compared to WT DMM mice 4 weeks after surgery. In addition, both ALN sham and DMM mice showed a reduced BS/BV compared to WT. 4 weeks after sham surgery Tb.Th. was highest in ALN mice. In DMM WT mice Tb.Sp. was higher compared to ALN and αCGRP-/−. 12 weeks after surgery (late OA stage), BS/BV of ALN sham mice was significantly reduced in relation to ALN DMM, WT and Tac1-/− sham, while Tb.Th. increased compared to WT. DMM significantly decreased Tb.N. and increased Tb.Sp. in Tac1-/− compared to sham 12 weeks after surgery. CTX-I concentrations were significantly higher in ALN compared to Tac1-/− mice 4 weeks after sham surgery. 12 weeks after sham surgery CTX-I concentrations of WT mice were increased compared to αCGRP-/− and Tac1-/− mice. Over time, DMM induced stronger changes in subchondral bone of WT mice compared to knockout strains. WT and αCGRP-/− sham mice also show alterations in bone parameters over time indicating age-related effects on bone structure. SP deficiency enhanced DMM-induced structural bone alterations in late stage OA emphasizing the importance of SP under pathophysiological conditions. Osteoclast inhibition with alendronate proved to be preservative for time-dependent changes of subchondral bone observed in both, DMM and sham mice. Interestingly, ALN treatment did not reduce bone turnover marker CTX-I, and additionally promoted early osteophyte formation in sham mice

Osteoarthritis (OA) is a joint degenerative disease leading to chronic pain and disability, thus resulting in a major socioeconomic health burden. OA, which has long been believed to be a cartilage disease, is now considered a whole-joint disorder affecting various anatomical structures, including subchondral bone. Hyaluronic Acid (HA) is commonly used as intra-articular viscosupplementation therapy for its mechanical features and biological effects. Bisphosphonates (BPs) are antiresorptive agents inhibiting recruitment and maturation of osteoclast precursors and activity of mature osteoclasts in the bone. Pre-clinical evidences in the literature, show that intra-articular BPs could impact on OA progression, slowing down or reversing it. The combination of HA biological and mechanical role and Alendronate (ALD) antiresorptive effect could be an interesting strategy for OA treatment. This study describes the synthesis and characterization of FID-134, a new chemical derivative of HA conjugated with ALD by means of a covalent bond, cleavable in physiological condition. FID-134 was synthesized starting from 500 kDa HA: chemical structure and functionalization degree with ALD were investigated by NMR and ICP-OES. Kinetics of ALD release from FID-134 was determined in TRIS buffer at 37°C and compared to a simple mixture of HA+ALD. 20mg/mL formulations of FID-134 and HA+ALD were investigated for viscoelastic properties, in absence and presence of Ca. 2+. ions. The cytotoxicity of FID-134 and free ALD were tested on Saos-2 osteoblasts (ATCC HTB-85) and on primary bovine chondrocytes (PBC) at day 1, 3 and 7. The efficacy of FID-134 was assessed in an inflammatory arthritis in vitro model, where bovine cartilage biopsies were exposed to IL-1β/OSM (10ng/mL) for 3 weeks; at the same time, cartilage explants were treated with FID-134. Collagen release in the surnatants was quantified and compared to controls. FID-134 structure was confirmed by NMR and the 20% mol/mol functionalization degree was determined by ICP-OES. Only about 50% of total bound ALD was released from FID-134 within 7 days, resulting slower compared to HA+ALD mixture. In presence of Ca. 2+. ions, viscoelastic properties of FID-134 dramatically improved, while HA+ALD formulation remained unaffected. The cytotoxicity of ALD was evident at 100 μM on Saos-2 and PBC after 3 days, while no cytotoxicity was observed at 7 days with FID-134. In the cartilage explant model, a strong collagen release was detected in inflammatory conditions after 3 weeks; this tendency was reversed, and collagen release halved when FID-134 was added to the biopsies. The synthesized HA-ALD adduct, FID-134, opens the door for a new approach for OA treatment. The results suggest that FID-134 could be beneficial in cartilage degradation and in restoration of subchondral bone function. Finally, local administration and controlled BP release would likely overcome the drawbacks of ALD oral administration, such as unspecific features and long-term toxic side effects

Introduction. Facet joint osteoarthritis (FJOA) is a prominent clinical hallmark of degenerative spine disorders. During disease progression, cartilage and subchondral bone tissues undergo increased turnover and remodeling. The structural changes to the subchondral tissue of FJOA have not been studied thus far. In this study, we performed a micro computed tomography (µCT) study of the subchondral cortical plate (SCP) and trabecular bone (STB) in FJOA and determined osteoarthritis-specific alterations. Methods. Twenty-four patients (11 male, 13 female, median age 65) scheduled for decompression and stabilization surgery for degenerative spinal stenosis were included in this study. FJOA specimens were harvested during surgery and analyzed by µCT. Bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N) were evaluated using CT Analyser. Lumbar facet joints without chondropathy from cadaveric specimens (9 male, 6 female, median age 57) served as healthy controls. Age-, gender- and disease-specific effects were identified by ANOVA (p<0.05) and significant differences confirmed by Bonferroni's post-test. Association between age and structural parameters was determined using correlation analysis. Results. Cortical and trabecular bone structural parameters of FJOA were similar between males and females. Compared to healthy controls, FJOA specimens demonstrated significantly greater trabecular Tb.N (1.97±0.11 vs 1.24±0.04 mm-1) and decrease of Tb.Sp (0.44±0.03 vs 0.69±0.03 mm). Conversely, subchondral cortical plate thickness (0.62±0.08 vs 1.60±0.08 mm) and porosity (22.9±1.9 vs 31.5±2.1%) were significantly less compared to healthy specimens. Tb.Th was equal between patients and controls. Age was positively correlated with Tb.N (r=0.48, p=0.02) and negatively correlated with Tb.Sp (r=−0.44, p=0.03) and cortical plate thickness (r=−0.52, p=0.04) in FJOA. Cortical and trabecular bone parameters did not associate in healthy and osteoarthritic facet joints. Conclusion. FJOA bone remodeling is characterized by thinning of the SCP and an increase in the number of subchondral trabeculae. Remodeling of cortical and trabecular bone might occur in an uncoupled fashion. Targeting elevated subchondral bone remodeling might slow progression of lumbar FJOA

There is no optimal therapy to stop or cure chondral degeneration in osteoarthritis (OA). Beside cartilage, subchondral bone is involved. The often sclerotic bone is mechanically less solid which in turn influences negatively chondral quality. Microfracturing as therapeutic technique aims to enhance bone quality but is applied only in smaller cartilage lesions. The osteoproliferative properties of Magnesium (Mg) have been shown repeatedly. 1-3. The present study examined the influence of micro-scaled Mg cylinders compared to sole drilling in an OA model. Ten New Zealand White rabbits underwent anterior crucial ligament transection. During 12 weeks after surgery, the animals developed OA as previously described. 4. In a second surgery, half of the animals received 20 drill holes (ø 0.5mm) and the other half received 20 drill holes, which were additionally filled with one Mg cylinder each. Extracapsular plication was performed in all animals. During the follow-up of 8 weeks three µ-computed tomographic (µCT) scans were performed: immediately after surgery and after four and eight weeks. Changes of bone volume, trabecular thickness and bone density were calculated and compared. µCT evaluation showed an increase in bone volume and trabecular thickness in both groups. This increase was significantly higher in rabbits which received Mg cylinders showing thrice as high values for both parameters (bone volume: Mg group +44.5%, drilling group +15.1%, p≤0.025; trabecular thickness: Mg group +53.2%, drilling group +16.9%, p≤0.025). Also bone density increased in both groups, but on a distinctly lower level and with no significant difference. Although profound higher bone volume was found after implantation of Mg cylinders, µCT showed similar levels of bone density indicating adequate bone quality in this OA model. Macroscopic and histological evaluation of cartilage condition have to reveal possible impact on OA progression. Additionally, current examination implement different alloys and influence on lameness

Knee osteoarthritis (OA) affects an estimated 250 million people worldwide, with a cure yet to be found. Consequently, there is an urgent need to improve our understanding of OA physiopathology. While knee OA has long been mostly described as a loss of cartilage thickness (CTh) and research has focused on this characteristic, the role of bone alterations is rapidly gaining in interest. Analyzing subchondral bone mineral density (sBMD) is particularly interesting because this could inform on the mechanical environment at the knee. However, there is a paucity of data on sBMD in literature mainly because of the lack of prior methods to measure this parameter. A method for 3D sBMD assessment based on computed tomography (CT) scans was recently proposed, thus allowing testing for sBMD differences in knee OA. This study aimed at comparing non-OA and medial OA knees in terms of tibial sBMD and CTh. Specifically, it was hypothesized that sBMD and CTh differ with OA. Ten knees with severe medial OA and 10 matched non-OA knees were analyzed after ethical approval (50% male; 60 ± 3 years old). The arthro-CT scans of the 20 knees were segmented using custom software to build 3D mesh models of the tibial bone and cartilage. CTh maps were obtained by calculating the distance between cartilage and bone meshes, while sBMD maps were calculated based on the intensity of the CT in the first 3mm of bone. For each knee, the average CTh and sBMD values over the entire medial and lateral compartments were calculated and used to determine the medial-to-lateral (M/L) CTh and sBMD ratios. Unpaired t-tests and receiver operating characteristic (ROC) were used for statistical analysis. The M/L sBMD ratio was significantly higher in OA compared to non-OA knees (1.14 ± 0.04 vs. 1.08 ± 0.03; p<0.01), whereas the CTh ratio was not significantly different between groups (0.70 ± 0.21 vs. 0.85 ± 0.10; p=0.06). No significant differences were found between OA and non-OA knees for the average medial CTh and sBMD (p>0.4). High classification performance was obtained for the sBMD ratio and low performance for the average sBMD in the medial compartment (areas under the ROC curve of 0.9 and 0.6, respectively). CTh ratio and medial compartment average provided medium classification performances (areas under the curve of 0.7). This study showed that sBMD differed between non-OA and severe medial OA knees and that sBMD M/L ratio was more sensitive to OA severity than CTh variables. These results brought new insights into the pathogenesis of knee OA, by supporting the idea that sBMD is altered with OA and suggesting that sBMD could play a role in disease development. Indeed, the mechanical stresses on the cartilages are related to the mechanical characteristics of the bones. Indirectly, this study also demonstrated the value of arthro-CT scans to simultaneously assess sBMD and CTh. Additional studies with larger cohorts of patients at different stages of the disease are necessary to better understand when changes in sBMD occur

Articular cartilage (AC) and subchondral bone (SB) are intimately intertwined, forming a complex unit called the AC-SB interface. Our recent studies have shown that cartilage and bone marrow are connected by a three-dimensional network of microchannels (i.e. cartilage-bone marrow microchannel connector; CMMC), which differ microarchitecturally in number, size and morphology depending on the maturation stage of the bone and the region of the joint. However, the pathological significance of CMMC is largely unknown. Here, we quantitatively assessed how CMMC microarchitecture relates to cartilage condition and regional differences in early idiopathic osteoarthritis (OA). Two groups of cadaveric female human femoral heads (intact cartilage vs early cartilage lesions) were identified and biopsy-based high-resolution micro-CT imaging was used. Subchondral bone (SB) thickness, CMMC number, maximum and minimum CMMC size, and CMMC morphology were quantified and compared between the two groups. The effect of joint region and cartilage condition on each dependent variable was examined. The number and morphology of CMMCs were influenced by the region of the joint, but not by the cartilage condition. On the other hand, the minimum and maximum CMMC size was modified by both joint location and cartilage condition. The smallest CMMCs were consistently found in the load bearing region (LBR) of the joint. Compared to healthy subjects, the size of the microchannels was increased in early OA, most notably in the non-load bearing region (NLBR) and the peripheral rim (PR) of the femoral head. In addition, subchondral bone thinning was observed in early OA as a localized event associated with areas of partial chondral defect. Our data suggest an enlargement of the SB microchannel network and a collective structural deterioration of the SB in early idiopathic OA

Osteoarthritis (OA) affects the whole joint and leads to chronic pain. The sympathetic nervous system (SNS) seems to be involved in OA pathogenesis, as indicated by in vitro studies as well as by our latest work demonstrating that sympathectomy in mice results in increased subchondral bone volume in the OA knee joint. We assume that chronic stress may lead to opposite effects, such as an increased bone loss in OA due to an elevated sympathetic tone. Therefore, we analyzed experimental OA progression in mice exposed to chronic stress. OA was induced in male C57BL/6J mice by surgical destabilization of the medial meniscus (DMM) and Sham as well as non-operated mice served as controls. Half of these groups were exposed to chronic unpredictable mild stress (CUMS). After 12 weeks, chronic stress efficiency was assessed using behavioral tests. In addition to measuring body weight and length, changes in subchondral bone were analyzed by μCT. Dynamic Weight Bearing system was used to monitor OA-related pain. Histological scoring will be conducted to investigate the severity cartilage degeneration and synovial inflammation. CUMS resulted in increased anxiety and significant decrease in body weight gain in all CUMS groups compared to non-CUMS groups. CUMS also increased serum corticosterone in healthy mice, with even higher levels in CUMS mice after DMM surgery. CUMS had no significant effect on subchondral bone, but subarticular bone mineral density and trabecular thickness were increased. Moreover, CUMS resulted in significant potentiation of DMM-associated pain. Our results suggest that the autonomic imbalance with increased sympathetic nervous activity induced by chronic stress exacerbates the severity of OA pain perception. We expect significantly increased cartilage degeneration as well as more severe synovial inflammation in CUMS DMM mice compared to DMM mice

TGF-β/Smad2 signaling is considered to be one of the important pathways involved in osteoarthritis (OA) and protein phosphatase magnesium-dependent 1A (PPM1A) functions as an exclusive phosphatase of Smad2 and regulates TGF-β signaling, here, we investigated the functional role of PPM1A in OA pathogenesis. PPM1A expressions in both human OA cartilage and experimental OA mice chondrocytes were analyzed immunohistochemically. Besides, the mRNA and protein expression of PPM1A induced by IL-1β treatment were also detected by q-PCR and immunofluorescence in vitro. OA was induced in PPM1A knockout (KO) mice by destabilization of the medial meniscus (DMM), and histopathological examination was performed. OA was also induced in wild-type (WT) mice, which were then treated with an intra-articular injection of a selective PPM1A inhibitor for 8 weeks. PPM1A protein expressions were increased in both human OA cartilage and experimental OA mice chondrocytes. We also found that treatment with IL-1β in mouse primary chondrocytes significantly increased both mRNA and protein expression of PPM1A in vitro. Importantly, our data showed that PPM1A deletion could substantially protect against surgically induced OA. Concretely, the average OARSI score and quantification of BV/TV of subchondral bone in KO mice were significantly lower than that in WT mice 8 weeks after DMM surgery. Besides, TUNEL staining revealed a significant decrease in apoptotic chondrocytes in PPM1A-KO mice with DMM operation. With OA induction, the rates of chondrocytes positive for Mmp-13 and Adamts-5 in KO mice were also significantly lower than those in WT mice. Moreover, compared with WT mice, the phosphorylation of Smad2 in chondrocytes was increased in KO mice underwent DMM surgery. However, articular-injection with SD-208, a selective inhibitor of TGF-β/Smad2 signaling could significantly abolish the chondroprotective phenotypes in PPM1A-KO mice. Additionally, both cartilage degeneration and subchondral bone subchondral bone sclerosis in DMM model were blunted following intra-articular injection with BC-21, a small-molecule inhibitor for PPM1A. Our study demonstrated that PPM1A inhibition attenuates OA by regulating TGF-β/Smad2 signaling. Furthermore, PPM1A is a potential target for OA treatment and BC-21 may be employed as alternative therapeutic agents for the management of OA

Abstract. OBJECTIVE. Changes in subchondral bone are one of few disease characteristics to correlate with pain in OA. 1. Profound neuroplasticity and nociceptor sprouting is displayed within osteoarthritic (OA) subchondral bone and is associated with pain and pathology. 2. The cause of these neural changes remains unestablished. Correct innervation patterns are indispensable for bone growth, homeostasis, and repair. Axon guidance signalling factor, Sema3A is essential for the correct innervation patterning of bony tissues. 3. , expressed in osteocytes. 4. and known to be downregulated in bone OA mechanical loading. 5. Bioinformatic analysis has also shown Sema3a as a differentially expressed pathway by bone in human OA patients. 6. HYPOTHESIS: Pathological mechanical load and inflammation of bone causes dysregulation of Sema3A signalling leading to perturbed sensory nerve plasticity and pain. METHODS. Human KOLF2-C1 iPSC derived nociceptors were generated by TALEN-mediated insertion of transcription factors NGN2+Brn3A and modified chambers differentiation protocol to produce nociceptor-like cells. Nociceptor phenotype was confirmed by immunocytochemistry. Human Y201-MSC cells were embedded in 3D type-I collagen gels (0.05 × 106 cell/gel), in 48-well plates and silicone plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) and soluble IL-6 receptor (sIL-6r (40ng/ml), IL6/sIL6r and mechanical load mimetic Yoda1 (5μM) or unstimulated (n=5/group) (48-well plates) or were mechanically loaded in silicone plates (5000μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). Conditioned media transfer was performed from osteocyte to nociceptor cultures assessed by continuous 24-hour phase contrast confocal microscopy. 24-hours after stimulation RNA was quantified by RT-qPCR (IL6) or RNAseq whole transcriptome analysis/DEseq2 analysis (Load). Protein release was quantified by ELISA. Normally distributed data with homogenous variances was analysed by two-tailed t test. RESULTS. IPSC-derived nociceptor-like cells display elongated (>5mm) dendritic projections and nociceptive molecular markers such as TUJ1, PrPH and Neun and TrkA. Sema3A signalling ligands were expressed in 100% of osteocyte cultures. Mechanical loading regulated the Sema3 pathway; Sema3A (0.4-fold, p<0.001), Sema3B (13-fold, p<0.001), Sema3C (0.4-fold, p<0.001). Under inflammatory stimulation by IL6/IL6sR, SEMA3A (7-fold, p=0.01) and receptor Plexin1 (3-fold, p=0.03) show significant regulation. Sema3A protein release showed a significant downregulation of Sema3A release by IL6/sIL6r+Yoda1 (2-fold, p=0.02). Continuous 24-hour phase contrast confocal microscopy measuring the number of extending/retreating dendritic projections revealed that sensory nerve cultures exposed to media from osteocytes stimulated with IL-6/sIL-6R+Yoda1 displayed significantly more invading dendritic projections (p=0.0175, 12-fold±SEM 3.5) across 3 random fields of view within a single stimulated neural culture and significantly fewer retracting dendritic projections (p=0.0075, 2-fold±SEM 0.33) compared to controls. CONCLUSIONS. Here we show osteocytic regulation of Sema3A under pathological mechanical loading and the ability of media pathologically loaded osteocyte cultures to induce the branching and invasion of cultured nociceptor-like cells as displayed in OA subchondral bone. 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