Gabriele Lehmann

Advanced glycation end product modification of bone proteins and bone remodelling: hypothesis and preliminary immunohistochemical findings

Background: The process of bone remodelling is disturbed in the development of osteoporosis. Objective: To investigate if proteins in osteoporotic bone are modified by advanced glycation end products (AGEs), and whether these alterations are related to measures of bone remodelling based on histomorphometric findings. Methods: Bone specimens taken from the iliac crest by bone biopsy of eight osteoporotic patients were investigated by histomorphometry and by immunohistochemical staining with the AGEs imidazolone and Nε-carboxymethyllysine. Results: Both AGEs were found in all bone specimens. The intensity of staining correlated with patient age. The percentage of bone surface covered with osteoblasts showed a significantly negative correlation with the staining intensity of both AGEs. Conclusions: It is known that AGEs can regulate proliferation and differentiation of osteoblastic cells and that AGE-specific binding sites are present in cultured osteoblast-like cells. Moreover, AGE induced biological effects in these cells might be mediated by RAGE (receptor of AGE) or by other AGE receptors in different stages of osteoblast development. The inverse relation between AGE staining intensity and the percentage of bone surface covered with osteoblasts in the trabecular bone may provide evidence that AGE modification of bone proteins disturbs bone remodelling.

Cytokines, Osteoprotegerin, and RANKL In Vitro and Histomorphometric Indices of Bone Turnover in Patients With Different Bone Diseases†

Cytokines are supposed to play an essential role in the regulation of the bone metabolic unit. However, information on cytokine production of primary human osteoblasts from patients with metabolic bone disease is scarce, and few attempts have been made to correlate such data to histomorphometric parameters of individual patients. We investigated 11 patients with metabolic bone disease referred to our outpatient department for bone biopsy and analyzed interleukin (IL)‐1, IL‐6, and TNF‐α protein release and gene expression in primary osteoblast cultures. Compared with four controls, five patients showed normal cytokine protein release, whereas six patients showed much higher levels of interleukin‐6 (26‐fold) and TNF‐α (84‐fold). All three cytokines were strongly correlated concerning gene expression and/or protein levels (r = 0.72–0.96). Histomorphometric analysis of the bone samples showed that eroded surface (ES/BS) as a parameter of bone resorption was significantly associated with TNF‐α. In addition, RANKL gene expression was positively associated with ES/BS and osteoclast surface (Oc.S/BS). Finally, the formation parameters osteoid volume and osteoid surface were negatively associated with TNF‐α. In conclusion, in an in vitro‐ex vivo model of bone cells obtained from a group of 11 patients with different forms of metabolic bone disease, cytokine release in conditioned medium was significantly associated with bone resorption and bone formation, as quantified by histomorphometry. TNF‐α seemed to be the more important cytokine; its effect on bone resorption could be mediated by RANKL.

Peripheral bone status in rheumatoid arthritis evaluated by digital X-ray radiogrammetry and compared with multisite quantitative ultrasound

The development of secondary osteoporosis in rheumatoid arthritis (RA) has recently become well recognized, characterized by demineralization at axial and in particular periarticular peripheral bone sites. Our aim was to evaluate multisite quantitative ultrasound (QUS) compared to digital X-ray radiogrammetry (DXR) by the quantification of cortical bone loss dependent on the severity of RA. Fifty-three patients with verified RA underwent QUS measurements (Sunlight Omnisense 7000) with estimation of the speed of sound (QUS-SOS) at the distal radius and at the phalanx of the third digit. Also, bone mineral density (DXR-BMD) and metacarpal index (DXR-MCI) were estimated on metacarpals II-IV using DXR technology. Additionally, Larsen score and Steinbroker stage were assessed. Disease activity of RA was estimated by disease activity score 28 (DAS 28). For the group with minor disease activity (3.2 ≤ DAS ≤ 5.1), QUS-SOS (phalanx) showed a significant association to DXR-BMD (R = 0.66) and DXR-MCI (R = 0.52). In the case of accentuated disease activity (DAS > 5.1), QUS-SOS of the radius revealed a significant correlation to DXR-BMD (R = 0.71) and DXR-MCI (R = 0.84), whereas for QUS-SOS (phalanx) no significant association to the DXR parameters was shown. The DXR parameters and, to a lesser extent, the QUS data also demonstrated pronounced declines in the case of accentuated disease activity (DAS > 5.1). Both DXR-BMD (−25.9 %, P < 0.01) and DXR-MCI (−38.6 %, P < 0.01) revealed a notable reduction dependent on the severity of RA. Otherwise, QUS-SOS marginally decreased, with −2.6% (radius) and −3.9% (phalanx). DXR revealed a significant reduction of DXR-BMD as well as DXR-MCI dependent on the severity of RA and surpassed multisite QUS as a promising diagnostic tool.

Influence of Image-Capturing Parameters on Digital X-Ray Radiogrammetry

The purpose of this study was to evaluate the importance of different image-capturing conditions, which might influence the characteristics of radiographs and, consequently, impact calculations of bone mineral density (BMD) and Metacarpal Index (MCI) using digital X-ray radiogrammetry (DXR). Radiographs of the left hand of deceased males were acquired three times using systematically varied parameters: 4-8 miliamp seconds (mA); 40-52 kV; film-focus distance (FFD); 90-130 cm; film sensitivity, 200/400; and different image modalities (conventional vs original digital radiographs as well as digital printouts). Furthermore, the interradiograph reproducibility using both conventional equipment and printouts vs originals of digital images and the intraradiograph reproducibility (either conventional or digital printouts) were evaluated. All BMD and MCI measurements were obtained with the DXR technology. The interradiograph reproducibility of DXR-BMD using conventional images under standardized conditions (6 mAs; 42 kV; 1 m FFD; film sensitivity of 200) was calculated to be coefficient of variation (CV) = 0.49% for Agfa Curix film and CV = 0.33% for Kodak T-MAT-Plus film, whereas reproducibility error using digital images ranged from CV = 0.57% (digital printouts; Philips) to CV = 1.50% (original digital images; Siemens). The intraradiograph reproducibility error was observed to be CV = 0.13% (conventional; Kodak film) vs CV = 0.27% (digital printouts; Philips). The BMD calculation was not noticeably affected by changes of FFD, exposure level, or film sensitivity/film brand, but was influenced by tube voltage (CV = 0.99% for Kodak film to CV = 2.05% for Siemens digital printouts). No significant differences were observed between the BMD and MCI data. DXR provides measurements of MCI and BMD with high precision and reproducibility. The measurements are unaffected by all tested image-capturing conditions, with the exception of tube voltage. In addition, different digital image devices clearly have an effect on DXR reproducibility.

Computerized digital imaging techniques provided by digital X-ray radiogrammetry as new diagnostic tool in rheumatoid arthritis.

PurposeOur study evaluates digital x-ray radiogrammetry (DXR) and Radiogrammetry Kit (RK) as a new diagnostic method for the measurement of disease-related osteoporosis including quantification of joint space narrowing dependent on the severity of rheumatoid arthritis (RA).Materials and MethodsA total of 172 unselected patients with RA underwent computerized measurements of bone mineral density (BMD) and metacarpal index (MCI) by DXR, as well as a semiautomated measurement of joint space distances at the metacarpal–phalangeal articulation (JSD-MCP 2–5), both were analyzed from plain radiographs of the nondominant hand.ResultsCorrelations between DXR-BMD and DXR-MCI vs. parameters of RK were all significant (0.34 < R < 0.61; p < 0.01). An expected negative association was observed between RK parameters and the different scoring methods (−0.27 < R < −0.59). The maximum relative decrease in BMD vs. MCI as measured by DXR between the highest and lowest RA severity group was −27.7% vs. −27.5% (p < 0.01) for the modified Larsen Score, whereas the minimal value of relative DXR-BMD and DXR-MCI reduction could be documented for the Sharp Erosion Score (−20.8% vs. −26.8%; p < 0.01). The relative reduction of mean JSD-MCP using RK significantly varied from −25.0% (Sharp Erosion Score) to −41.2% (modified Larsen Score). In addition, an excellent reproducibility of DXR and RK could be verified.ConclusionDXR in combination with RK could be a promising, widely available diagnostic tool to supplement the different scoring methods of RA with quantitative data, allowing an earlier and improved diagnosis and more precision in determining disease progression.

Value of digital X-ray radiogrammetry in the assessment of inflammatory bone loss in rheumatoid arthritis

Introduction Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by progressive joint destruction based on the synovitis of large and small joints (1,2). The small joints of the hands and feet are particularly affected in the majority of RA patients, leading to destruction of articular tissue, including cartilage and bone (2). Osteoporosis, a typical phenomenon of RA, occurs in two forms: periarticular osteoporosis, adjacent to the inflamed joints, and generalized osteoporosis, resulting in an increased risk of fractures. Periarticular osteoporosis is a specific feature of bone involvement in RA (3,4) and is one of the bone involvement criteria in addition to erosions in the American College of Rheumatology criteria for RA (5). The onset of periarticular bone loss in the hands and feet is an early sign of RA (6) and may precede erosions (7). Periarticular bone loss is mainly caused by the local release of inflammatory agents and is a direct consequence of the inflammatory process (8), whereas generalized bone loss is additionally influenced by immobility and treatment effects (e.g., glucocorticoids) (9–11). In the hand, inflammatory bone loss includes not only the direct periarticular region of the joints, but also the cortical bone of the metacarpal diaphysis, indicating that inflammation-induced bone loss is present independent of direct synovial contact (12–14). Immunologic aspects of periarticular bone loss in RA

Treatment of osteoporosis after liver transplantation with ibandronate.

Osteoporosis is a major side‐effect after liver transplantation (LTX). Therefore, the objective of the study was to evaluate the efficacy of ibandronate to reduce fractures after LTX.  Seventy‐four patients after LTX were included in the study and measurements of bone mineral density (BMD) of lumbar spine and proximal femur using dual energy X‐ray absorptiometry (DEXA) were performed prior to and 3, 6, 12 and 24 months after surgery. The study group (IBA) consisted of 34 patients who received calcium (1 g/day), vitamin D3 (800–1000 IE/day) and ibandronate 2 mg every 3 months intravenously for 1 year. The control group consisted of 40 patients (CON) who received calcium and vitamin D3 at the same dosages. Prevalence of new fractures was predefined as primary endpoint. Changes of BMD and biochemical markers of bone metabolism were also investigated. In all patients, we found a reduction of BMD in the first few months after LTX. In the lumbar spine and the proximal femur the maximum reduction occurred 3 and 6 months post‐LTX. One and 2 years after transplantation, the group receiving ibandronate demonstrated a better recovery from loss of BMD and a significantly lower prevalence of fractures (IBA 2 vs. CON 10 P < 0.04, χ2). Ibandronate with calcium and vitamin D3 reduces the BMD‐loss after LTX and decreases the rate of bone fractures significantly.

Temperature influence on DXA measurements: bone mineral density acquisition in frozen and thawed human femora

BackgroundDetermining bone mineral density (BMD) with dual-energy x-ray absorptiometry (DXA) is an established and widely used method that is also applied prior to biomechanical testing. However, DXA is affected by a number of factors. In order to delay decompositional processes, human specimens for biomechanical studies are usually stored at about -20°C; similarly, bone mineral density measurements are usually performed in the frozen state. The aim of our study was to investigate the influence of bone temperature on the measured bone mineral density.MethodsUsing DXA, bone mineral density measurements were taken in 19 fresh-frozen human femora, in the frozen and the thawed state. Water was used to mimic the missing soft tissue around the specimens. Measurements were taken with the specimens in standardized internal rotation. Total-BMD and single-BMD values of different regions of interest were used for evaluation.ResultsFourteen of the 19 specimens showed a decrease in BMD after thawing. The measured total-BMD of the frozen specimens was significantly (1.4%) higher than the measured BMD of the thawed specimens.ConclusionBased on our findings we recommend that the measurement of bone density, for example prior to biomechanical testing, should be standardized to thawed or frozen specimens. Temperature should not be changed during measurements. When using score systems for data interpretation (e.g. T- or Z-score), BMD measurements should be performed only on thawed specimens.

Joint damage in rheumatoid arthritis: assessment of a new scoring method

IntroductionThe aim of this study was to assess a novel approach for the quantification of finger joint space narrowing and joint destruction in patients with rheumatoid arthritis (RA) focusing on the peripheral hand articulations.MethodsA total of 280 patients with verified RA underwent computerized semi-automated measurements of joint space distance at the finger articulations based on radiographs. The Z-Score, which can differentiate between joint space alterations caused by RA versus age/gender-related changes, was calculated as a comparative parameter. The severity of joint space narrowing was also quantified by the Sharp Score. Sensitivity and specificity of the Z-Score (based on joint space widths differentiated for each peripheral finger joint) were evaluated to reveal the potential for the occurrence of erosions. Additionally, the potential of the Z-Score regarding the differentiation of therapeutic effects on joint space widths in patients under a therapy of methotrexate versus leflunomide was performed.ResultsThe Z-Scores of finger articulations in patients with RA were generally decreased. Metacarpal-phalangeal (MCP) joint articulations showed a continuous significant decline of -1.65 ± 0.30 standard deviations dependent on the Sharp Score. The proximal-interphalangeal joints also revealed a significant reduction of the Z-Score (-0.96 ± 0.31 standard deviations). The sensitivity and specificity of MCP joint space distance for the detection of erosions were 85.4% versus 55.2%. The Sharp Score for joint space narrowing was not able to detect different treatments, whereas an accentuated stabilization of joint space narrowing could be identified for the Z-Score of the MCP joints in patients treated with leflunomide and methotrexate.ConclusionThe Z-Scoring method based on computer-aided analysis of joint space widths was able to reliably quantify severity-dependent joint space narrowing in RA patients. In the future, calculation of a Z-Score based on gender-specific and age-specific reference data shows the potential for a surrogate marker of RA progression that comprehends the early identification of patients with RA, and in particular those with erosive course of the disease, enabling a timely therapeutic strategy for cartilage protection.

The usefulness of computer-aided joint space analysis in the assessment of rheumatoid arthritis

OBJECTIVE Computer-aided joint space analysis (CAJSA) is a newly developed technique for the measurement of radiogeometrically detectable joint space widths of the metacarpal-phalangeal (JSD-MCP) and proximal-interphalangeal articulations (JSD-PIP). The aim of this study was to verify the sensitivity and specificity of these CAJSA measurements in the assessment of established RA. METHODS Four hundred and fifty-eight participants (248 healthy subjects, 210 RA patients) underwent computerized semi-automated measurements of the JSD-MCP and JSD-PIP articulations (CAJSA, Radiogrammetry Kit, Version 1.3.6) based on digitally performed radiographs. The Sharp joint space narrowing score was also performed to determine RA-related joint space narrowing. RESULTS The significant severity-dependent reduction for JSD-MCP was -44.0% and for JSD-PIP, -25.94% between Sharp scores 0 and 3. The sensitivity and specificity of JSD-MCP (total) was 88.1% versus 77.8%, respectively (AUC = 0.920; P < 0.001). Furthermore, JSD-PIP (total) revealed a lower sensitivity and specificity with 61.4% and 88.7% (AUC = 0.878; P < 0.001). CONCLUSION The CAJSA method presented a reliable assessment of disease-related joint space narrowing in patients suffering from RA with excellent sensitivity and specificity. By providing quantitative data, other scoring methods could be significantly improved, and thereby the accuracy of the diagnosis and a better therapeutic evaluation could be achieved.