Impact of the biopsy forceps size on histological analysis and performances of the histological scoring systems.

To improve the reliability of the quantitative scorings of the synovial biopsies, we evaluate whether diameter of arthroscopic forceps influences histological quality of synovial tissue and/or histological scores and we compare the intra- and inter-observer performances of the main histological scoring systems. Synovial biopsies were retrieved in the same part of the joint using 1, 2 and 4 mm diameters grasping forceps. After standard staining and immunohistochemistry with anti-CD68 antibody, slides were scored blindly by 2 independent experienced operators for tissue quality and with Krenn score, de Bois-Tak score and CD68 semi-quantitative score. Four samples did not pass quality control. No difference other than a higher number of vessels in the 4 mm versus 2 mm forceps (p = 0.01) was found among the 3 groups. CD68 score was significantly higher in the 2 versus 4 mm forceps (p = 0.009). So we concluded that only vessels quantification and CD68 semi-quantitative score seemed affected by the forceps size. The intra-reader agreement was variable across observers and features: 0.78 (0.66-0.87) for the Krenn scoring system, 0.89 (0.78-0.97) for the de Bois-Tak score and 0.93 (0.81-1.00) for the CD68 score. Interobserver reliabilities of Krenn score, de Bois-Tak score and CD68 scores were satisfactory: 0.95 (0.92-0.99) for Krenn, 0.98 (0.96-0.99) for de Bois-Tak and 0.80 (0.71-0.89) for CD68.

New Proteins Contributing to Immune Cell Infiltration and Pannus Formation of Synovial Membrane from Arthritis Diseases.

An inflamed synovial membrane plays a major role in joint destruction and is characterized by immune cells infiltration and fibroblast proliferation. This proteomic study considers the inflammatory process at the molecular level by analyzing synovial biopsies presenting a histological inflammatory continuum throughout different arthritis joint diseases. Knee synovial biopsies were obtained from osteoarthritis (OA; n = 9), chronic pyrophosphate arthropathy (CPPA; n = 7) or rheumatoid arthritis (RA; n = 8) patients. The histological inflammatory score was determined using a semi-quantitative scale based on synovial hyperplasia, lymphocytes, plasmocytes, neutrophils and macrophages infiltration. Proteomic analysis was performed by liquid chromatography-mass spectrometry (LC-MS/MS). Differentially expressed proteins were confirmed by immunohistochemistry. Out of the 1871 proteins identified and quantified by LC-MS/MS, 10 proteins (LAP3, MANF, LCP1, CTSZ, PTPRC, DNAJB11, EML4, SCARA5, EIF3K, C1orf123) were differentially expressed in the synovial membrane of at least one of the three disease groups (RA, OA and CPPA). Significant increased expression of the seven first proteins was detected in RA and correlated to the histological inflammatory score. Proteomics is therefore a powerful tool that provides a molecular pattern to the classical histology usually applied for synovitis characterization. Except for LCP1, CTSZ and PTPRC, all proteins have never been described in human synovitis.

Proteins involved in the endoplasmic reticulum stress are modulated in synovitis of osteoarthritis, chronic pyrophosphate arthropathy and rheumatoid arthritis, and correlate with the histological inflammatory score.

It is now well recognized that osteoarthritis (OA) synovial membrane presents inflammatory components. The aim of this work is to provide evidence that similar inflammatory mechanisms exist in synovial membrane (n = 24) obtained from three pathologies presenting altogether an inflammatory gradient: OA, chronic pyrophosphate arthropathy (CPPA) and rheumatoid arthritis (RA). Synovial biopsies were first characterized by a histological score based on synovial hyperplasia and infiltration of lymphocytes, plasma cells, polymorphonuclear and macrophages. All biopsies were also analyzed by 2D-nano-UPLC-ESI-Q-Orbitrap for protein identification and quantification. Protein levels were correlated with the histological score. Histological score was in the range of 3 to 8 for OA, 5 to 13 for CPPA and 12 to 17 for RA. Of the 4,336 proteins identified by mass spectrometry, 51 proteins were selected for their strong correlation (p < 0.001) with the histological score of which 11 proteins (DNAJB11, CALR, ERP29, GANAB, HSP90B1, HSPA1A, HSPA5, HYOU1, LMAN1, PDIA4, and TXNDC5) were involved in the endoplasmic reticulum (ER) stress. Protein levels of S100A8 and S100A9 were significantly higher in RA compared to OA (for both) or to CPPA (for S100A8 only) and also significantly correlated with the histological score. Eighteen complement component proteins were identified, but only C1QB and C1QBP were weakly correlated with the histological score. This study highlights the inflammatory gradient existing between OA, CPPA and RA synovitis either at the protein level or at the histological level. Inflamed synovitis was characterized by the overexpression of ER stress proteins.

Did Osteoblastic Cell Therapy Improve the Prognosis of Pre-fracture Osteonecrosis of the Femoral Head? A Randomized, Controlled Trial.

BACKGROUND: In patients with nontraumatic osteonecrosis of the femoral head (ONFH), implantation of bone marrow aspirate concentrate (BMAC) could delay the progression of osteonecrosis and improve symptoms in pre-fracture ONFH. However, the BMAC content, especially in osteoblastic stem cells, could have an important individual variability. An autologous osteoblastic cell product could improve the effect of such cell-based therapy. QUESTIONS/PURPOSES: (1) Does autologous osteoblastic cell therapy decrease the likelihood of progression to subchondral fracture with or without early collapse corresponding to Association Research Circulation Osseous (ARCO) classification Stage III or higher, and provide a clinically important pain improvement compared with BMAC treatment alone? (2) Were patients treated with osteoblastic cell therapy less likely to undergo subsequent THA? (3) What proportion of patients in the treatment and control groups experienced adverse events after surgery? METHODS: Between 2004 and 2011, we treated 279 patients for Stage I to II hip osteonecrosis (ON) with surgery. During that time, our general indications for surgery in this setting included non-fracture ON lesions. To be eligible for this randomized, single-blind trial, patients needed to have an ONFH Stage I or II; we excluded those with traumatic ONFH, hemoglobinopathies and positive serology for hepatitis B, C or HIV. Of those treated surgically for this diagnosis during the study period, 24% (67) agreed to participate in this randomized trial. Hips with pre-fracture ONFH were randomly treated with a core decompression procedure associated with either implantation of a BMAC (BMAC group; n = 26) or osteoblastic cell (osteoblastic cell group; n = 30). The groups were not different in terms of clinical and imaging characteristics. The primary study outcome was treatment response, defined as the absence of progression to subchondral fracture stage (ARCO stage III or higher) plus a clinically important pain improvement defined as 1 cm on a 10-cm VAS. The secondary endpoint of interest was the frequency in each group of subsequent THA and the frequency of adverse events. The follow-up duration was 36 months. We used an as-treated analysis (rather than intention-to-treat) for our efficacy endpoint, and an intention-to-treat analysis for adverse events. Overall, 26 of 26 patients in the BMAC group and 27 of 30 in the osteoblastic cell group completed the trial. RESULTS: At 36 months, no clinically important differences were found in any study endpoint. There was no difference in the proportion of patients who had progressed to fracture (ARCO stage III or higher; 46% of the BMAC hips [12 of 26] versus 22% in the hips with osteoblastic cells [six of 27], hazard ratio, 0.47 [95% CI 0.17 to 1.31]; p = 0.15). There was no clinically important difference in VAS pain scores. No differences were found for either the WOMAC or the Lequesne indexes. With the numbers available, there was no difference in the proportion of patients in the groups who underwent THA at 36 months 15% (four of 27) with osteoblastic cells versus 35% (nine of 26) with BMAC; p = 0.09 With the numbers available, we found no differences between the treatment and control groups in terms of the frequencies of major adverse events. CONCLUSIONS: We found no benefit to osteoblastic cells over BMAC in patients with pre-collapse ONFH; side effects were uncommon and generally mild in both groups. This study could be used as pilot data to help determine sample sizes for larger (presumably multicenter) randomized controlled trials. However, this novel treatment cannot be recommended in routine practice until future, larger studies demonstrate efficacy. LEVEL OF EVIDENCE: Level II, therapeutic study.

Inefficacy of autologous bone marrow concentrate in stage three osteonecrosis: a randomized controlled double-blind trial.

PURPOSE: The fracture stage of non-traumatic osteonecrosis (ON stage 3) of the femoral head (ONFH) has an unfavourable prognosis frequently requiring total hip replacement (THR). The percentage could be lowered after core decompression. In earlier non-fracture ON stages, implantation of autologous bone marrow aspirate concentrate (BMAC) improved the effect of core decompression. The purpose was to evaluate the effect of BMAC in addition to core decompression in stage 3 ONFH. METHODS: A double blind RCT was conducted comparing two groups: core decompression plus saline injection or core decompression plus BMAC implantation. Both patients and assessors were blinded to the treatment assignments. Evaluations were done at baseline, three, six, 12, and 24 months, including pain (VAS), WOMAC, side-effects, radiological evolution including ARCO subclassifications, together with possible THR requirement. The primary endpoint was the need for THR. The second endpoints included the clinical symptoms such as pain and functional ability and the progression of the ON lesions as well as the appearance of osteoarthritis features (ARCO stage 4). Both groups included 23 hips (19 patients). RESULTS: No differences were found between the groups for THR requirements, clinical tests, and radiological evolution. In both groups, 15/23 hips needed THR. The radiological evolution of the ONFH lesions in term of location, extension, surface collapse, and dome depression was moderate in both groups and was not correlated with the need of THR. CONCLUSIONS: Implantation of BMAC after core decompression did not produce any improvement of the evolution of ONFH stage 3. Level of evidence I.

Non traumatic osteonecrosis of the femoral head is associated with low bone mass.

OBJECTIVE: Osteoporosis (OP) and osteonecrosis of the femoral head (ONFH) share common clinical and pathophysiological features we sought to determine whether ONFH was associated with an increased prevalence of OP and whether the increased prevalence of OP was related to the stage of ONFH at diagnosis. METHODS: We included 243 patients with ONFH and 399 age and sex-matched healthy controls. Data was gathered including demography, risk factors, ARCO staging of ONFH and bone mineral density (BMD). RESULTS: Overall, BMD (defined by the T-score) was significantly lower in the ONFH group at both the femoral head (-0.96±1.11) and the lumbar spine (-1.22±1.47) compared to the control group (-0.55±0.97 and -0.73±1.31) (p<0.01). The ONFH group depicted a significantly higher proportion of osteopenia (50.39% vs 40.87%, p=0.027) and of OP (18.78% vs 7.33%, p<0.001) relative to the control group. Stage 1 and 2 ONFH patients (53.86%, p=0.0203; OR=1.54 (95% CI: [1.04; 2.29])) were at a higher risk of osteopenia than the control group (40.88%), but not stages 3 or 4 (48.47%, p=0.2569; OR=1.27 (95% CI: [0.78; 2.06]). Patients with stage 3 or 4 ONFH (25.31%, p<0.001; OR=3.93 (95% CI: [1.63; 10.96])) were at a higher risk of osteoporosis than patients in the stage 1 and 2 ONFH (7.24%), and compared to the control group (7.33%, adj. p-value<0.001; OR=4.89 (95% CI: [2.77; 8.76]). CONCLUSIONS: Non-traumatic osteonecrosis of the femoral heads is associated with low bone mineral density. This study showed that fractural stages ONFH were associated with a 5-fold risk of osteoporosis.

Biomarkers of inflammation and innate immunity in atrophic nonunion fracture.

BACKGROUND: Nonunion is a failure of healing following a bone fracture. Its physiopathology remains partially unclear and the discovery of new mediators could promote the understanding of bone healing. METHODS: Thirty-three atrophic nonunion (NU) patients that failed to demonstrate any radiographic improvement for 6 consecutive months were recruited for providing serum samples. Thirty-five healthy volunteers (HV) served as the control group. Proteomics studies were performed using SELDI-TOF-MS and 2D-DIGE approaches, associated or not with Proteominer® preprocessing, to highlight biomarkers specific to atrophic nonunion pathology. Peak intensities were analyzed by two statistical approaches, a nonparametric Mann-Whitney U tests (univariate approach) and a machine-learning algorithm called extra-trees (multivariate approach). Validation of highlighted biomarkers was performed by alternative approaches such as microfluidic LC-MS/MS, nephelometry, western blotting or ELISA assays. RESULTS: From the 35 HV and 33 NU crude serum samples and Proteominer® eluates, 136 spectra were collected by SELDI-TOF-MS using CM10 and IMAC-Cu(2+) ProteinChip arrays, and 665 peaks were integrated for extra-trees multivariate analysis. Accordingly, seven biomarkers and several variants were identified as potential NU biomarkers. Their levels of expression were found to be down- or up-regulated in serum of HV vs NU. These biomarkers are inter-α-trypsin inhibitor H4, hepcidin, S100A8, S100A9, glycated hemoglobin ß subunit, PACAP related peptide, complement C3 α-chain. 2D-DIGE experiment allowed to detect 14 biomarkers as being down- or up-regulated in serum of HV vs NU including a cleaved fragment of apolipoprotein A-IV, apolipoprotein E, complement C3 and C6. Several biomarkers such as hepcidin, complement C6, S100A9, apolipoprotein E, complement C3 and C4 were confirmed by an alternative approach as being up-regulated in serum of NU patients compared to HV controls. CONCLUSION: Two proteomics approaches were used to identify new biomarkers up- or down-regulated in the nonunion pathology, which are involved in bone turn-over, inflammation, innate immunity, glycation and lipid metabolisms. High expression of hepcidin or S100A8/S100A9 by myeloid cells and the presence of advanced glycation end products and complement factors could be the result of a longstanding inflammatory process. Blocking macrophage activation and/or TLR4 receptor could accelerate healing of fractured bone in at-risk patients.

A prospective cohort study of the clinical presentation of non-traumatic osteonecrosis of the femoral head: spine and knee symptoms as clinical presentation of hip osteonecrosis.

PURPOSE: To study the clinical presentation of femoral head osteonecrosis (ONFH). Publications dedicated to this aspect of ONFH are rare. Our aim was to systematically collect and describe the clinical data. METHODS: A prospective survey was conducted in a cohort of ONFH recruited from a dedicated clinic for osteonecrosis. The history of symptoms, medical management, and physical findings were obtained from 88 patients suffering from 125 ONFH. Subgroups were formed: bilateral versus unilateral ONFH, radiological stages 1-2 (pre-fractured) versus fractured stage 3 versus stage 4. RESULTS: ONFH was bilateral in 63 %, especially in corticosteroid users and in sickle-cell cases. These patients were younger but had similar BMIs compared to the unilateral cases. The pain was mechanical in 79 % of hips and inflammatory in 21 %. Acute pain at the onset was present in 55 % of hips. The localization of this pain was variable, including in the groin, the buttocks, or diffused in the lower limbs. A limp was present in 50 % of the patients, only when one hip was painful. The physical examination of the hip was normal in 31 %, especially in stages 1-2 (55 %). The diagnosis delay was 12 months, with inadequate medical management in 51 % of patients. CONCLUSIONS: In ONFH cases, no typical clinical pattern was found. The clinical presentation was very variable, sometimes having spine or knee symptoms with a normal physical examination of the hip. ONFH should be systematically suspected in cases of onset of pain in the pelvis, buttocks, groin, and lower limbs.

The apoptosis of osteoblasts and osteocytes in femoral head osteonecrosis: its specificity and its distribution.

The pathogenesis of nontraumatic osteonecrosis (ON) remains unclear. Some studies have suggested that nontraumatic ON is attributed to increased osteocytic apoptosis. To test this hypothesis, a controlled study must compare the apoptosis of osteocytes and osteoblasts in cases of ON and osteoarthritis (OA). To assess either the localized or diffuse patterns of this increased osteocytic and osteoblastic apoptosis, we evaluated both the proximal and distal regions of necrotic areas. Femoral heads resected for total hip prosthesis were included for this study. Of these, 10 were ON cases-three were induced by corticosteroids, three by alcohol abuse, one resulted from trauma, one resulted from hyperlipemia, and two were idiopathic-10 were osteoarthritis cases, and 1 from a patient suffering from a subcapital fracture. The TUNEL reaction was used to detect the apoptosis in osteoblasts and osteocytes. A semi-quantitative evaluation was conducted, at both distal and proximal areas relative to the lesions, specifically in the area surrounding the necrotic region in the osteonecrosis cases, in the eburnated bone in the osteoarthritis cases, and in the subchondral bone fracture. The apoptosis of osteoblasts and osteocytes was statistically more frequent in the regions close to the necrotic areas in the ON group. No difference was found in the unpaired areas. In the ON group, no difference was found in terms of the etiological factors. During ON, the apoptosis of osteocytes and osteoblasts is increased proximally to the necrotic regions in the patients presenting with osteoarthritis and subcapital fractures. This increase was found not only in the corticosteroid-induced ON cases but also in the idiopathic and alcohol abuse- and trauma-induced ON cases.

(111)Indium-oxine labelling for evaluating the homing process of autologous osteoblasts implanted percutaneously in atrophic nonunion fractures.

PURPOSE: The aim of the study was to control the in vivo localisation of implanted cells in cell-based therapies. Labelling cells with (111)indium-oxine is one of the most interesting methods proposed. We evaluated this method in the setting of autologous osteoblast implantation in nonunion fractures. METHODS: An in vitro study of osteoblasts was conducted after (111)indium-oxine labelling. Radioactivity retention and viability, proliferation and the ability to produce alkaline phosphatase were evaluated in a seven-day culture. In vivo labelling of implanted osteoblastic cells was conducted during a therapeutic trial of atrophic nonunion fractures, with the leakage outside the nonunion site and local uptake evolution at four, 24 and 48 hour being studied. RESULTS: The mean labelling efficiency for osteoprogenitors was 78.8 ± 4.6 %. The intracellular retention was 89.4 ± 2.1 % at three hours and 67.3 ± 4.7 % at 18 hours. The viability assessed at three hours was 93.7 ± 0.6 %. After seven days of culture, morphology and alkaline phosphatase staining were similar for both labelled and unlabelled control cells, although the proliferation rate was decreased in the labelled cells. Some local intraosseous leakage was observed in four of 17 cases. All patients showed uptake at the injection site, with four having no other uptake. Four patients showed additional uptake in the bladder, liver and spleen, while 11 patients had additional uptake in the lungs in addition to the bladder, liver and spleen. The activity ratios (injection site/body) were 48 ± 28 % at four hours, 40 ± 25 % at 24 hours and 35 ± 25 % at 48 hours. After correcting for decay, the activity within the injection site was 82 ± 15 % at 24 hours and 69 ± 11 % at 48 hours compared with the activity measured at four hours. No relationship was found between uptake and radiological bone repair. CONCLUSIONS: The (111)indium-oxine labelling appears to be a good method for monitoring the behaviour of the osteoblastic cells after their implantation in atrophic nonunion fractures.

Intra-articular use of a medical device composed of hyaluronic acid and chondroitin sulfate (Structovial CS): effects on clinical, ultrasonographic and biological parameters.

BACKGROUND: This pilot open noncontrolled study was designed to assess the efficacy of intra-articular injections of a solution combining hyaluronic acid (HA) and chondroitin sulphate (CS) in the treatment of outpatients affected by knee osteoarthrosis. FINDINGS: Thirty patients with knee OA have been included. The primary objective was to assess clinical efficacy as measured by pain and Lequesne's index. Secondary objectives were to assess potential effect of the treatment on ultrasound parameters, safety and biomarkers of cartilage metabolism and joint inflammation. After a selection visit (V1), the study treatment was administered 3 times on a weekly basis (V2, V3, V4). Follow-up was planned 6 (V5) and 12 weeks (V6) after the first intra-articular injection. Efficacy results showed a reduction in mean pain at V3 and V6 and in functional impairment, the most marked changes being measured at the two follow-up visits (V5 and V6). Although statistical significance was not achieved due to small sample size, a clear tendency towards improvement was detectable for ultrasound assessments as well as biomarkers. Except for a mild injection site hematoma for which the drug causal relationship could not be excluded, no adverse effect of clinical relevance was recorded during the study. CONCLUSION: Although this pilot study was performed according to an open design only, the ultrasound as well as biomarkers changes strongly suggest a non-placebo effect. These preliminary results call now for a randomized controlled study to confirm the clinical relevance of the observed results. TRIAL REGISTRATION: #ISRCTN91883031.

Autologous bone marrow cell implantation in the treatment of non-traumatic osteonecrosis of the femoral head: Five year follow-up of a prospective controlled study.

OBJECTIVE: To determine the efficacy of bone marrow cell implantation into the necrotic lesion of the femoral head on clinical symptoms and the progression of osteonecrosis of the femoral head in comparison with core decompression. METHODS: We studied nineteen patients and twenty four hips with early stage osteonecrosis of the femoral head. The hips were allocated to either core decompression only or core decompression and implantation of bone marrow cells. Both patients and assessors were blind with respect to treatment group assignment. The primary outcomes were clinical symptoms and disease progression. RESULTS: Bone marrow implantation afforded a significant reduction in pain and in joint symptoms and reduced the incidence of fractural stages. At 60 months, eight of the eleven hips in the control group had deteriorated to the fractural stage whereas only three of the thirteen hips in the bone marrow graft group had progressed to that stage. Survival analysis showed a significant difference in the time to failure between the two groups at 60 months. Patients had only minor side-effects after the treatments. CONCLUSIONS: This long term follow-up study confirmed that implantation of autologous bone marrow cells in the necrotic lesion might be an effective treatment for patients with early stages of osteonecrosis of the femoral head.

Discovery and biochemical characterisation of four novel biomarkers for osteoarthritis.

OBJECTIVE: Knee osteoarthritis (OA) is a heterogeneous, complex joint pathology of unknown aetiology. Biomarkers have been widely used to investigate OA but currently available biomarkers lack specificity and sensitivity. Therefore, novel biomarkers are needed to better understand the pathophysiological processes of OA initiation and progression. METHODS: Surface enhanced laser desorption/ionisation-time of flight-mass spectrometry proteomic technique was used to analyse protein expression levels in 284 serum samples from patients with knee OA classified according to Kellgren and Lawrence (K&L) score (0-4). OA serum samples were also compared to serum samples provided by healthy individuals (negative control subjects; NC; n=36) and rheumatoid arthritis (RA) patients (n=25). Proteins that gave similar signal in all K&L groups of OA patients were ignored, whereas proteins with increased or decreased levels of expression were selected for further studies. RESULTS: Two proteins were found to be expressed at higher levels in sera of OA patients at all four K&L scores compared to NC and RA, and were identified as V65 vitronectin fragment and C3fpeptide. Of the two remaining proteins, one showed increased expression (unknown protein at m/z of 3762) and the other (identified as connective tissue-activating peptide III protein) was decreased in K&L scores >2 subsets compared to NC, RA and K&L scores 0 or 1 subsets. CONCLUSION: The authors detected four unexpected biomarkers (V65 vitronectin fragment, C3f peptide, CTAP-III and m/z 3762 protein) that could be relevant in the pathophysiological process of OA as having significant correlation with parameters reflecting local inflammation and bone remodelling, as well as decrease in cartilage turnover.

Phases 1-3 clinical trials using adult stem cells in osteonecrosis and nonunion fractures.

NONUNION FRACTURES AND ASEPTIC BONE NECROSIS ARE TWO PATHOLOGICAL CONDITIONS HAVING SOME IMPAIRMENT OF THE CELLULAR PART OF THE REPAIR: a reduction of MSC and of the osteoblastic activation. Both are good candidates for cell-based therapies using stem cells. We made a review of the published human trials. Only autologous bone marrow aspirate implantation was until now used. In Nonunion, a direct injection-15 to 150 ml-was made in 4 case series studies. In another, the bone marrow aspirate was concentrated before injection. The results were good. In bone necrosis, only one level 1 study was published. The results at 24 months were positive in terms of reduction of the necrosis and appearance of collapse. In 3 case series studies, a treatment with concentrated bone marrow aspirates was deemed useful with good results in 76 to 96%. These results are interesting but need confirmation by controlled studies.

Cellular-based therapy for osteonecrosis.

This review article describes bone remodeling in the context of osteonecrosis as a bone disease, the use of stem cells in bone and vascular diseases, and cellular therapy in osteonecrosis.

Contrast-enhanced coded phase-inversion harmonic sonography of knee synovitis correlates with histological vessel density: 2 automated digital quantifications.

OBJECTIVE: To use contrast-enhanced coded phase-inversion harmonic B-mode sonography to assess the acoustic enhancement of the synovial area of the knee; and to compare the data with the histological vessel density. METHODS: Eleven patients eligible for a knee arthroscopy were studied. Acoustic quantification was carried out by a digital image analysis program that detects the time-dependent increase [intensity (time) = k x time + C] of gray-level intensity in all the pixels of a specific region of interest (ROI) following intravenous injection of the microbubble contrast agent sulfur hexafluoride. Echo-guided synovial biopsies were carried out in the same ROI. Synovial vessel areas were quantified after Factor VIII immunostaining of synovial biopsies using an automated digital image analysis. RESULTS: Significant (p < 0.05) correlations were observed between histological vessel density and percentage of the synovial area with a k value > 0.01 (r = 0.93) and k(max) values (r = 0.79), as well as between the 2 latter parameters (r = 0.72). The histological vessel density and the 2 acoustic parameters were also significantly correlated with the logarithm of erythrocyte sedimentation rate (r = 0.77, r = 0.87, r = 0.67, respectively) and with log C-reactive protein serum concentration (r = 0.69, r = 0.83, r = 0.62, respectively). CONCLUSION: Contrast-enhanced coded phase-inversion harmonic B-mode sonography coupled with an appropriate data analysis method is a new tool to identify and quantify vessel density in knee synovitis.

Inefficacy of the cementation of femoral head collapse in glucocorticoid-induced osteonecrosis.

The objective of this paper was to assess the efficacy of cementation of the femoral head in stage III glucocorticoid-induced osteonecrosis. Ten hips (nine patients) were treated by the injection of low-viscosity cement to reduce the collapse. The follow up included clinical and radiological assessments preoperatively and at 3, 6 and 12 months after surgery. The visual analogue scale (VAS) score, the Lequesne index and the Western Ontario and McMaster University Osteoarthritis Index (WOMAC) score did not show any significant improvement. Eight of the ten hips showed a worsening of the collapse and required total hip arthroplasty during follow up. The mean time before total hip replacement was 8.6 +/- 7 months. The other two hips did not show any relapse of collapse nor functional worsening at the maximum follow up of 5 years. Our results suggest that cement injection is not a treatment that should be proposed for glucocorticoid-induced osteonecrosis.

Peroxisome proliferator-activated receptor-gamma1 is dephosphorylated and degraded during BAY 11-7085-induced synovial fibroblast apoptosis.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a central role in whole body metabolism by regulating adipocyte differentiation and energy storage. Recently, however, PPAR-gamma has also been demonstrated to affect proliferation, differentiation, and apoptosis of different cell types. As we have previously shown that BAY 11-7085-induced synovial fibroblast apoptosis is prevented by PPAR-gamma agonist 15d-PGJ2; the expression of PPAR-gamma in these cells was studied. Both PPAR-gamma1 and PPAR-gamma2 isoforms were cloned from synovial fibroblast RNA, but only PPAR-gamma1 was detected by Western blot, showing constitutive nuclear expression. Within minutes of BAY 11-7085 treatment, a PPAR-gamma1-specific band was shifted into a form of higher mobility, suggesting dephosphorylation, as confirmed by phosphatase treatment of cell extracts. Of interest, BAY 11-7085-induced PPAR-gamma1 dephosphorylation was followed by PARP and caspase-8 cleavage as well as by PPAR-gamma1 protein degradation. PPAR-gamma1 dephosphorylation was followed by the loss of PPAR-DNA binding activity ubiquitously present in synovial fibroblast nuclear extracts. Unlike the phosphorylated form, dephosphorylated PPAR-gamma1 was found in insoluble membrane cell fraction and was not ubiquitinated before degradation. PPAR-gamma1 dephosphorylation coincided with ERK1/2 phosphorylation that accompanies BAY 11-7085-induced synovial fibroblasts apoptosis. 15d-PGJ2, PGD2, and partially UO126, down-regulated ERK1/2 phosphorylation, protected cells from BAY 11-7085-induced apoptosis, and reversed both PPAR-gamma dephosphorylation and degradation. Furthermore, PPAR-gamma antagonist BADGE induced PPAR-gamma1 degradation, ERK1/2 phosphorylation, and synovial fibroblasts apoptosis. The results presented suggest an anti-apoptotic role for PPAR-gamma1 in synovial fibroblasts. Since apoptotic marker PARP is cleaved after PPAR-gamma1 dephosphorylation but before PPAR-gamma1 degradation, dephosphorylation event might be enough to mediate BAY 11-7085-induced apoptosis in synovial fibroblasts.