Connective tissue growth factor (CTGF/CCN2) in haemophilic arthropathy and arthrofibrosis: a histological analysis.

INTRODUCTION: Joint haemorrhage is the principal clinical manifestation of haemophilia frequently leading to advanced arthropathy and arthrofibrosis, resulting in severe disability. The degree and prevalence of arthrofibrosis in hemophilic arthropathy is more severe than in other forms of arthropathy. Expression of connective tissue growth factor (CTGF) has been linked to many fibrotic diseases, but has not been studied in the context of haemophilic arthropathy. AIM: We aim to compare synovial tissues histologically from haemophilia and osteoarthritis patients with advanced arthropathy in order to compare expression of proteins that are possibly aetiologic in the development of arthrofibrosis. METHODS: Human synovial tissues were obtained from 10 haemophilia and 10 osteoarthritis patients undergoing joint surgery and processed for histology and immunohistochemistry. RESULTS: All samples from haemophilia patients had synovitis with hypertrophy and hyperplasia of synovial villi. Histologically, synovial tissues contained hyperplastic villi with increased cellularity and abundant haemosiderin- and ferritin-pigmented macrophage-like cells (HMCs), with a perivascular localization in the sub-surface layer. CTGF staining was observed in the surface layer and sub-surface layer in all haemophilia patients, exclusively co-localizing with HMCs. Quantification showed that the extent of CTGF-positive areas was correlated with the degree of detection of HMCs. CTGF was not observed in any of the samples from osteoarthritis patients. CONCLUSION: Using histological analysis, we showed that CTGF expression is elevated in haemophilia patients with arthrofibrosis and absent in patients with osteoarthritis. Additionally, we found that CTGF is always associated with haemosiderin-pigmented macrophage-like cells, which suggests that CTGF is produced by synovial A cells following the uptake of blood breakdown products.

Ankle fusion in patients with haemophilia.

Ankle fusion in patients with haemophilia is a well-accepted treatment for end-stage arthropathy. However, current published outcome data are based on small sample sizes and generally short-term follow-up. The aim of this study was to evaluate the long-term results of ankle fusion in a large group of haemophilic patients treated at a single institution. The results of 57 ankle fusions performed on 45 patients between 1971 and 2010 were reviewed retrospectively. Data were gathered for type and severity of haemophilia, HIV status, fixation technique, postoperative complications and requirement of additional surgeries. A modified American Orthopaedic Foot & Ankle Society (AOFAS) hindfoot score was calculated for 20 ankles available for follow-up. Patients were followed for a mean of 6.6 years. There were no intra-operative or immediate postoperative complications related to fusion of the ankle. The overall non-union rate was 10.4% for tibio-talar fusion and 8.3% for sub-talar fusion. This rate was reduced to 3.7% and 5.6%, respectively, after the introduction of newer surgical techniques in 1995. None of these non-unions required revision surgery. The modified AOFAS scale demonstrated that 75% had no pain in the operated ankle a mean of 7.2 years following surgery. The remaining 25% scored their average pain as 3 of 10. The functional portion of the score suggested that patients have good alignment, minimal activity limitations or gait abnormalities, and can walk long distances. We conclude that ankle fusion successfully relieves pain and provides a good functional outcome. It is an appropriate treatment for end-stage haemophilic arthropathy of the ankle.

Opinions on radiosynovectomy for chronic haemophilic synovitis: point/counterpoint.

Joint bleeding is the hallmark of severe haemophilia and the major cause of disability in patients with this coagulopathy. Repeated bleeding into the same joint can lead to chronic synovitis and progressive arthropathy. Radiosynovectomy is one option for the treatment of chronic haemophilic synovitis, but concerns about the risks of exposure to ionizing radiation have divided clinicians as to the safety and appropriate use of the procedure. This article presents two differing viewpoints, one from a pair of orthopaedic surgeons who collectively have performed more than 300 radiosynovectomies in patients with haemophilia. They maintain that radiosynovectomy is a simple, effective, safe and low-cost technique children and adults with chronic haemophilic synovitis. The other perspective is from an experienced haemophilia treater who directs a major US haemophilia treatment centre. She believes that unresolved questions about the safety of radiation exposure in children argue against the use of radiosynovectomy in paediatric patients with haemophilia.

Inter- and intra-observer reliability of radiographic scores commonly used for the evaluation of haemophilic arthropathy.

Although the severity of haemophilic arthropathy is commonly assessed using established radiographic scoring systems, there is limited available information about their inter- and intra-observer reliability. The purpose of the present study was to establish the inter-observer reliability (IEOR) and intra-observer reliability (IAOR) of three different methods available for the classification of haemophilic arthropathy, including the Arnold and Hilgartner classification, a modification to the Arnold and Hilgartner system described by Luck et al., and the classification described by Pettersson et al. Antero-posterior and lateral radiographs of 54 haemophilic joints were included for the analysis. To determine the IEOR for each one of the three radiographic systems, the radiographs were randomly evaluated by four observers, including two orthopaedic surgeons, one orthopaedic resident and one haematologist. For the determination of IAOR, all four reviewers repeated the assessment in a similar fashion, after a period of at least 2 weeks. IEOR and IAOR for the three classification systems was established using kappa (kappa) statistics. A Spearman rank correlation was used to determine the similarities between each reviewer's own interpretative scales. The IEOR was low for the Arnold and Hilgartner system (kappa = 0.35, P < or = 0.001) and the Luck system (kappa = 0.38, P < or = 0.001), but even lower for the Pettersson system (kappa = 0.06, P = 0.1). For the Pettersson system, particularly low kappa values were observed for the presence or absence of osteoporosis (kappa = 0.11, P = 0.0027), enlarged epiphysis (kappa = 0.10, P = 0.0039), erosion of joint margins (kappa = 0.11, P = 0.0018), and joint deformity (kappa = 0.16, P = 0.00001). However, a relatively high Spearman rank correlation for all three scales [r(s) = 0.75 (P < 0.001) for Arnold and Hilgartner system, r(s) = 0.74 (P < 0.001) for the Luck system and r(s) = 0.81 (P < 0.001) for Pettersson system] indicated an overall, general agreement among the reviewers with regard to the severity of the haemophilic arthropathy. There was a moderate IAOR value for both, the Arnold and Hilgartner system (kappa = 0.57, P = 0.00001) and the Luck system (kappa = 0.62, P = 0.00001) with a low IAOR value for the Pettersson system [kappa = 0.22, P = 0.00001). Currently available radiographic scoring systems for haemophilic arthropathy have low inter- and intra-observer reliability rates. Improvements, either through education or modification of the scoring systems, are critical in an era where correlations between clinical and radiographic scores have received significant attention.

Phosphate-32 colloid radiosynovectomy in hemophilia: outcome of 125 procedures.

Radiosynovectomy offers a potentially effective, minimally invasive option for patients with chronic hemarthrosis and synovitis. The long-term outcome of patients with hemophilia who were treated with phosphate-32 chromic phosphate radiosynovectomy was evaluated. One hundred twenty-five procedures in 81 patients were done. Two- to 10-year followup by age and joint included joint bleeding and quality-of-life assessment. In addition, a relative cost comparison, scintigraphic imaging, and evaluation of biodistribution of the radionuclide were done. Of 125 procedures, 54% resulted in complete cessation of bleeding into the treated joint after the procedure, and 73% of patients reported improved mobility of the treated joint. Of patients 18 years old and younger, 79% had a greater than 75% reduction in bleeding incidence, and of patients older than 40 years, only 56% had a similar reduction. Seventy-nine percent of patients surveyed had a significant improvement in quality of life attributable to the treated joint. No evidence of significant leakage was observed. Billing records analysis indicated that radiosynovectomy costs less than 5% of surgical synovectomy. Phosphate-32 chromic phosphate radiosynovectomy is a clinically useful, safe, and cost-effective outpatient procedure in the treatment of patients with chronic hemarthrosis and synovitis.

32P chromic phosphate radiosynovectomy for chronic haemophilic synovitis.

The principal medical consequence of haemophilia is the development of arthropathy, initiated by a haemarthrosis giving rise to chronic synovitis. Traditional methods of synovectomy include open excision and arthroscopy each of which require substantial amounts of clotting factor concentrate for several weeks, and in the case of open synovectomy, is often associated with loss of range of motion and arthrofibrosis. Radiosynovectomy, the intra-articular injection of low penetration radiocolloids, has been utilized outside the United States for over 20 years. Since 1988, our centre has performed 170 radiosynovectomies utilizing 32P chromic phosphate (32P). This study reports results of 130 32P radiosyovectomies with an average follow-up of 36.5 months (6-140 months). For primary procedures, excellent and good results (haemarthrosis reduction from 75 to 100%) were obtained in 79.2% of cases at 6 months to 8 years. For repeat procedures a combination of excellent and good results were obtained in 62.4% of cases at 6 months to 3 years. Regression analysis showed no correlation between results and age or degree of arthropathy. Radiation was well contained within the joint. There were no observed or identified complications. The procedure is highly cost effective in comparison to open surgical or arthroscopic synovectomy.

Characteristics of degeneration in an unstable knee with a coronal surface step-off.

To investigate the effect of instability on the remodelling of a minor articular surface offset, we created a 0.5 mm coronal step-off of the medial femoral condyle in 12 New Zealand white rabbits and transected the anterior cruciate ligament (ACL). A control group of 12 rabbits had only ACL resection and the opposite knee was used as the non-operated control. The osteoarthritic changes at 6, 12 and 24 weeks after surgery were evaluated histologically. In addition, changes in the immunological detection of 3-B-3(-) and 7-D-4 chondroitin-6-sulphate epitopes were determined because of the previous association of such changes with repair of cartilage and early osteoarthritis. In the instability/step-off group there was rapidly progressing focal degeneration of cartilage on the high side of the defect, not seen in previous step-off studies in stable knees. The rest of the femoral condyles and the tibial plateaux of the instability/step-off group had moderate osteoarthritis similar to that of the instability group. 3-B-3(-) was detectable in the early and the intermediate stages of osteoarthritis but no staining was seen in the severely damaged cartilage zones. Immunoreactivity with 7-D-4 increased as degeneration progressed.

Homeobox B3, B4, and C6 gene product expression in osteosarcomas as detected by immunocytochemistry.

Osteosarcoma (OS) is a malignant neoplastic disease of the bone, of mesenchymal origin and with considerable morphologic heterogeneity, consisting of malignant stoma with evidence of malignant osteoid, bone and/or cartilage production. The mammalian homeobox (HOX) represents a highly conserved DNA motif of 183 base pairs, encoding the 61 amino acid DNA-binding homeodomain, through which the HOX gene products regulate the transcription of other genes involved in onto- and histogenesis. Re-expression of HOX proteins has been identified in a wide variety of neoplastically transformed cell types and it seems that the HOX genes represent yet another family of oncofetal antigens involved in both normal development and oncogenesis, as well as tumor tissue progression. During this study, the expression pattern of three HOX gene products (HOX-C6, -B3, and -B4) was examined immunocytochemically in human osteosarcoma (OS) tissues. In all observed (16/16) OS cases, HOX-C6 was present in over 90% of the neoplastically transformed cells (+4), demonstrating a high to medium grade (A to B) staining intensity. Similar results were obtained in OS cells for the other two observed proteins (HOX-B3 and -B4; over 90% or +4 and a high to medium grade staining intensity or A and B). The significance of the expression of class I HOX proteins in the pathobiology, diagnosis and prognostication of human OS should be established by further investigations.

Benign and malignant cartilage tumors of bone and joint: their anatomic and theoretical basis with an emphasis on radiology, pathology and clinical biology. II. Juxtacortical cartilage tumors.

In part I, we reviewed the varied clinical presentations, pathogenesis, histologic findings, radiologic findings, and treatment of intramedullary cartilaginous lesions of bone. In this section, we will evaluate our cases and consultations of juxtacortical cartilaginous tumors. Radiographic differential diagnosis includes the numerous juxtacortical lesions particularly osteochondroma, parosteal chondroma, Trevor's disease, trauma (fracture and periostitis ossificans), and the low- and high-grade surface osteosarcomas. By emphasizing pathogenesis in conjunction with radiographic and histologic findings, pitfalls in diagnosis and subsequent treatment can be avoided in such cases.

Cartilage changes caused by a coronal surface stepoff in a rabbit model.

Coronal stepoffs of 0.5 mm (equal to the cartilage height) were created on the medial femoral condyles of adult, skeletally mature rabbits as a model for articular surface incongruity. After 3, 6, 12, and 24 weeks, tissue was analyzed histologically using hematoxylin and eosin and Safranin O staining, autoradiographs were made of the femoral condyles, and immunohistologic analysis was done for 3-B-3(-) and 7-D-4 chondroitin sulfate epitopes. An overlapping flap from the high toward the low side and an increase of the cartilage height on the low side of the defect were observed as permanent features of adaptation throughout the entire followup. Significant degeneration was not seen around the lesion or in the tibial cartilage opposing a stepoff defect. Autoradiography showed a three-phase response to the lesion: an early increase in radiolabeled sulfate (35SO4) uptake, a sharp decline of 35SO4 uptake, and finally a late recovery of the autoradiographic signal indicating partial recovery of proteoglycan synthetic activity. After an early increase, immunohistologic analysis for 3-B-3(-) showed a subsiding tendency by 24 weeks, and the staining with 7-D-4 remained elevated uniformly in the vicinity of the lesion. A rabbit femoral stepoff defect with an offset of 0.5 mm may remodel and not lead to degeneration within the first 6 months after injury in a stable joint.

Capitated reimbursement for medical services returns control of the patient to the surgeon.

Limiting the spending on healthcare services is a societal necessity, whether externally budget-driven with reduced fee for service or salary, or internally controlled through prospective payment capitation. No reimbursement system is inherently good or bad. Ethical physicians will place patient well-being first and focus on the delivery of quality care, regardless of the payment method. There are several methods for the distribution of capitation payments to physicians, each with different levels of financial incentive to provide services. In one fully evolved embodiment of capitation, a payer carves out the entire orthopedic disease segment and contracts with an orthopedic organization for all musculoskeletal services within a defined geographic region. This form of capitation offers the advantage of returning control of patient care to the orthopedic surgeon.

Radiosynovectomy's clinical applications and cost effectiveness: a review.

It is apparent that from the work of the authors and many others, including the work of Rivard, Sledge, Zuckerman, among others, that radiosynovectomy has an important role to play in providing effective treatment of affected joints associated with rheumatoid arthritis and other forms of arthritis as well as the hemophiliac joint. The treatment offers relief from the effects of recurrent joint effusion with an approximately 60% to 66% favorable response and from recurrent hemarthrosis in the hemophiliac joint with an approximately 75% to 80% favorable response. The impact of providing radiosynovectomy as an alternative to surgical synovectomy is seen, where postoperative side effects such as joint stiffness are avoided, improved quality of life is repeatedly documented, and the cost savings in health care dollars, particularly evident in the hemophiliac joint in this relatively small population, are potentially enormous. With almost two million people in the United States suffering from rheumatoid arthritis, the potential savings in health care dollars is also enormous. As with any use of in vivo radiopharmaceuticals, the potential for radiation-induced damage exists. However, with a 25 plus year record of use, more optimally configured radiopharmaceuticals, and the addition of maneuvers to minimize potential joint leakage, the risk of radiation induced damage appears to be minimal. It appears as though radiosynovectomy is an effective as well as cost-effective alternative to surgical synovectomy and is becoming the procedure of choice particularly in the hemophiliac patient with recurrent hemarthrosis and synovitis who has failed medical therapy. It is also the procedure of choice in patients for whom surgery is contraindicated because of the presence of clotting factor inhibitors.

Clinical and prognostic significance of the expression of the c-erbB-2 and c-erbB-3 oncoproteins in primary and metastatic malignant melanomas and breast carcinomas.

Several growth factors and proto-oncogenes play a leading regulatory role during human carcinogenesis. In this systematic immunocytochemical study we observed the expression (overexpression) of the c-erbB-2 and c-erbB-3 oncoproteins in 30 primary cutaneous malignant melanomas (CMMs), 10 already metastasized malignant melanomas (MMMs) and 15 lymph-node negative breast carcinomas (BCs). Both oncoproteins were expressed as a result of either oncogene amplification or post-translational stabilization c-erbB-2 alone is unable to bind neuregulins, but it is able to act as a pan c-erbB receptor subunit. Heterodimerization between cerbB-2 and c-erbB-3 is required to initiate neuregulin directed signal transduction. We employed an indirect, four step streptavidinbiotin conjugated immunocytochemical technique for antigen detection. The visualization of the primary antigen-antibody reaction was carried out with alkaline phosphatase or immunoperoxidase labeling and the use of the appropriate enzymatic substrates. The presence of c-erbB-2 oncoprotein was detected in 12/30 CMMs, 8/10 MMMs and 6/15 BCs, while c-erbB-3 was identified in 14/30 CMMs, 7/10 MMMs and 6/15 BCs. The intensity of the cell membrane localized immunoreactivity was observed to be greater when the c-erbB-2 oncoprotein was targeted (A, AB and B). The c-erbB-3 oncoprotein was also detected in the cytoplasm with medium intensity (B, BC and C). Unfortunately, little is known concerning the range of oncoprotein overexpression after formalin fixation and paraffin embedding. We demonstrated overexpression localized to several cell clones within the oncoprotein positive population of malignant cells. The immunocytochemically defined extent of expression of both oncoproteins was between 10-40% (+ to +2) of the total cell population in the malignant melanomas and 20-35% (+2) of the total cell population in the BCs. In conclusion a) the results of the present study demonstrate the presence of c-erbB-2 and c-erbB-3 oncoprotein expression (overexpression) in melanoma and breast carcinoma, and b) oncogene receptor directed immunotherapy, as part of a more individualized anti-cancer treatment, represents a potentially valuable targeted treatment for the future.

Immunocytochemical detection of the p170 multidrug resistance (MDR) and the p53 tumor suppressor gene proteins in human breast cancer cells: clinical and therapeutical significance.

The phenomenon of multidrug resistance (MDR) is characterized by resistance to several unrelated cytotoxic agents, such as anthracyclines, vinca alkaloids and epipodophylline derivatives. MDR has been described frequently in human breast carcinoma (BC), as has the alteration of the p53 gene, responsible for ensuring the integrity of the genome. The most well known type of MDR is associated with the overexpression of a 170kD glycoprotein (p170). This mechanism of MDR is the result of increased transcription of the mdr 1 gene. The p170 glycoprotein in normal cells with excretory functions is a permanent component of a membrane transport system and an increase in its expression, such as that which occurs in neoplastically transformed cells, results in increased drug efflux and decreased intracellular drug concentration. The present immunocytochemical study was carried out on routine, formalin fixed, paraffin-wax embedded, 3-4 microns thick tissue sections of 15 breast carcinomas, treated at the University of Southern California. The immunoperoxidase antigen detection protocol, developed by Hsu et al (1981) was employed using three anti-p170 monoclonal antibodies (MoABs), JSB-1, C-219 and C-494 (Signet Laboratories, Dedham, MA, USA), and the anti-p53 MoAB PAb1801 (NeaMarkers, Inc., Fremont, CA, USA). 14/15 BCs contained large proportions of cells which displayed the characteristic transmembrane localized expression of p170. All 15 BCs were comprised of distinct groups of cells which accumulated p53 in their nucleus and occasionally in their cytoplasm. A distinct, heterogeneous immunophenotype (IP) of the cells comprising the tumor microenvironment and different grades of neoplastic differentiation was also observed. In 5/15 BC cases intense immunoreactivity, correlating with p170 overexpression, was detected. The 15 BCs exhibited different staining patterns typical for each anti-p170 MoAB. MoAB JSB-1 reacted strongly with the transmembranic antigen epitope, as did MoAB C-494, the long incubation time employed with MoAB C-219, on the other hand, resulted in inhomogeneous cytoplasmic staining. Previous reports suggest a direct correlation between the presence of the p170 glycoprotein in human cancer cells and the poor response to chemotherapy. Furthermore, the genetic instability, which is the consequence of the loss of wild-type p53 function, may be the underlying property which allows highly malignant cells to amplify the mdr 1 gene and thus become resistant to a wide spectrum of cytotoxic drugs.

Intraoperative pathologic consultation (IOC) for tumors of the bone.

When combining clinical examination, laboratory information and noninvasive imaging studies the differential diagnosis of bone lesions is narrowed. For those who are not experts in the field the major purpose of the IOC is to insure that adequate tissue has been obtained and to triage the tissue in the process preparing imprints, whenever possible; fixing some tissue for possible electron microscopic review; placing some tissue in B5 fixative for better cytologic detail; and to save some undecalcified tumor tissue in formalin in case immunostaining procedures are required. Most community pathologists should not be attempt to make an absolute diagnosis at the time of IOC, in many cases. The surgeon should always be warned that despite seeming benignancy 50% of primary bone tumors are malignant, that benign lesions can prove to be low grade sarcomas after full review, and vice versa that occasional cellular, "pleomorphic" lesions can be benign (aneurysmal bone cyst, early reparative and pseudosarcomatous lesions). Following review of the permanent sections, and other appropriate procedures an accurate diagnose is possible in the majority of cases. If the diagnosis is particularly difficult or questionable the above materials can be sent to a bone tumor specialist.

Immunophenotypic characterization of human primary and metastatic melanoma infiltrating leukocytes.

During a systematic screening of melanoma infiltrating mononuclear and polynuclear immunological effector cells we employed 10 well characterized monoclonal antibodies, directed against leukocyte differentiation antigens, and the alkaline phosphatase conjugated, indirect, streptavidinbiotin immunocytochemical antigen detection technique and when necessary, the recently developed antigen retrieval method to enhance the intensity of the immunoreactivity to characterize the heterogeneous infiltrate of human primary (n = 30) and metastatic (n = 10) melanomas. Our study is the first to employ formalin fixed, paraffin embedded tissue sections in seeking to determine the ex vivo presence of tumor infiltrating leukocytes, including T lymphocytes, in human melanomas. We established the presence of some type of melanoma infiltrating host's immunological effector cells in all 40 observed melanoma cases'. More specifically, we found NK cells, macrophages and granulocytes in 30 out of 30 PMs and 10 out of 10 MMs. These effector cells represented the vast majority (> 80%) of the melanoma infiltrating immunocompetent cells. T lymphocytes were observed in 20 out of 30 PMs and 6 out of 10 MMs, but their numbers represented only between 5% to 10% of the heterogeneous leukocytic infiltrate. B cells were found in 22 out of 30 PMs and 8 out 10 MMs, their numbers representing less than 5%. Presence of cells of the dendritic reticulum, involved in antigen presentation was not determined in any of the observed PMs and MMs. There were more than enough macrophages and neutrophils, cells which are deeply involved in antigen presentation and, as previously mentioned, we found these cells within the melanomas. Thus defective antigen presentation cannot be the reason for the small proportion of T lymphocytes. What is to blame is probably the increased dedifferentiation of the melanoma immunophenotype (IP) to a degree that MHC class I molecules and the antigens complexed with them are lost and therefore there is nothing to be recognized by the T lymphocytes. In view of these results it is our-opinion that following the initial non-specific immune reaction, which may not be tumor specific, the specific immune response of effector T cells takes over. After significant IP changes by the majority of melanoma cells, including the internalization or shedding of MHC class I antigens, these highly specialized cells are no longer effective and the immune system responds by causing the reappearance of NK cells, macrophages and granulocytes. Thus, the relationship between melanoma and the host's immune system is evolutionarily dynamic: changes are readily made by either side when necessitated.

Effects of valgus tibial angulation on cartilage degeneration in the rabbit knee.

Thirteen adult female rabbits underwent unilateral osteotomy of the proximal tibia. In nine animals, 30 degrees of valgus angulation was created; in four animals, osteotomy without angulation was performed. After a 12-week survival period, the knee joints were processed for histology by staining with hematoxylin and eosin and safranin O. Additionally, the chondroitin sulphate epitopes 3-B-3(-) and 7-D-4 were evaluated immunohistochemically as markers of osteoarthritis. Changes of the articular surface of the tibia were visualized by scanning electron microscopy. Light microscopic evaluation by the Mankin et al. scoring system revealed mild or moderate damage of the cartilage in the lateral compartment of angulated extremities when compared with the control side. Immunohistology with the monoclonal 3-B-3 and 7-D-4 antibodies showed no increased expression of these epitopes in the lateral compartments of the knee. Scanning electron microscopic evaluation of the tibial surfaces revealed slight surface damage localized to the central, weight-bearing portion of the lateral tibial plateau of angulated extremities. Angulation of 30 degrees led to only mild degenerative changes of the cartilage. These results indicate that, in the short term, cartilage has considerable capacity to withstand the effects caused by severe angulation of the limb.