Short-term perioperative parecoxib is not detrimental to shaft fracture healing in a rat model.

OBJECTIVES: Experimental studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) may have negative effects on fracture healing. This study aimed to assess the effect of immediate and delayed short-term administration of clinically relevant parecoxib doses and timing on fracture healing using an established animal fracture model. METHODS: A standardized closed tibia shaft fracture was induced and stabilized by reamed intramedullary nailing in 66 Wistar rats. A 'parecoxib immediate' (Pi) group received parecoxib (3.2 mg/kg bodyweight twice per day) on days 0, 1, and 2. A 'parecoxib delayed' (Pd) group received the same dose of parecoxib on days 3, 4, and 5. A control group received saline only. Fracture healing was evaluated by biomechanical tests, histomorphometry, and dual-energy x-ray absorptiometry (DXA) at four weeks. RESULTS: For ultimate bending moment, the median ratio between fractured and non-fractured tibia was 0.61 (interquartile range (IQR) 0.45 to 0.82) in the Pi group, 0.44 (IQR 0.42 to 0.52) in the Pd group, and 0.50 (IQR 0.41 to 0.75) in the control group (n = 44; p = 0.068). There were no differences between the groups for stiffness, energy, deflection, callus diameter, DXA measurements (n = 64), histomorphometrically osteoid/bone ratio, or callus area (n = 20). CONCLUSION: This study demonstrates no negative effect of immediate or delayed short-term administration of parecoxib on diaphyseal fracture healing in rats.Cite this article: G. A. Hjorthaug, E. Søreide, L. Nordsletten, J. E. Madsen, F. P. Reinholt, S. Niratisairak, S. Dimmen. Short-term perioperative parecoxib is not detrimental to shaft fracture healing in a rat model. Bone Joint Res 2019;8:472-480. DOI: 10.1302/2046-3758.810.BJR-2018-0341.R1.

2016 Comprehensive Update of the Banff Working Group on Liver Allograft Pathology: Introduction of Antibody-Mediated Rejection.

The Banff Working Group on Liver Allograft Pathology reviewed and discussed literature evidence regarding antibody-mediated liver allograft rejection at the 11th (Paris, France, June 5-10, 2011), 12th (Comandatuba, Brazil, August 19-23, 2013), and 13th (Vancouver, British Columbia, Canada, October 5-10, 2015) meetings of the Banff Conference on Allograft Pathology. Discussion continued online. The primary goal was to introduce guidelines and consensus criteria for the diagnosis of liver allograft antibody-mediated rejection and provide a comprehensive update of all Banff Schema recommendations. Included are new recommendations for complement component 4d tissue staining and interpretation, staging liver allograft fibrosis, and findings related to immunosuppression minimization. In an effort to create a single reference document, previous unchanged criteria are also included.

Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis.

Diabetic nephropathy and extracellular matrix.

Diabetic nephropathy (DN) is a serious complication in diabetes. Major typical morphological changes are the result of changes in the extracellular matrix (ECM). Thus, basement membranes are thickened and the glomerular mesangial matrix and the tubulointerstitial space are expanded, due to increased amounts of ECM. One important ECM component, the proteoglycans (PGs), shows a more complex pattern of changes in DN. PGs in basement membranes are decreased but increased in the mesangium and the tubulointerstitial space. The amounts and structures of heparan sulfate chains are changed, and such changes affect levels of growth factors regulating cell proliferation and ECM synthesis, with cell attachment affecting endothelial cells and podocytes. Enzymes modulating heparan sulfate structures, such as heparanase and sulfatases, are implicated in DN. Other enzyme classes also modulate ECM proteins and PGs, such as matrix metalloproteinases (MMPs) and serine proteases, such as plasminogen activator, as well as their corresponding inhibitors. The levels of these enzymes and inhibitors are changed in plasma and in the kidneys in DN. Several growth factors, signaling pathways, and hyperglycemia per se affect ECM synthesis and turnover in DN. Whether ECM components can be used as markers for early kidney changes is an important research topic, whereas at present, the clinical use remains to be established.

Coating of titanium with hydroxyapatite leads to decreased bone formation: A study in rabbits.

OBJECTIVES: An experimental rabbit model was used to test the null hypothesis, that there is no difference in new bone formation around uncoated titanium discs compared with coated titanium discs when implanted into the muscles of rabbits. METHODS: A total of three titanium discs with different surface and coating (1, porous coating; 2, porous coating + Bonemaster (Biomet); and 3, porous coating + plasma-sprayed hydroxyapatite) were implanted in 12 female rabbits. Six animals were killed after six weeks and the remaining six were killed after 12 weeks. The implants with surrounding tissues were embedded in methyl methacrylate and grinded sections were stained with Masson-Goldners trichrome and examined by light microscopy of coded sections. RESULTS: Small amounts of bone were observed scattered along the surface of five of the 12 implants coated with porous titanium, and around one out of 12 porous coated surfaces with Bonemaster. No bone formation could be detected around porous coated implants with plasma-sprayed hydroxyapatite. CONCLUSION: Porous titanium coating is to some degree osteoinductive in muscles.

Guidelines for the diagnosis of antibody-mediated rejection in pancreas allografts-updated Banff grading schema.

The first Banff proposal for the diagnosis of pancreas rejection (Am J Transplant 2008; 8: 237) dealt primarily with the diagnosis of acute T-cell-mediated rejection (ACMR), while only tentatively addressing issues pertaining to antibody-mediated rejection (AMR). This document presents comprehensive guidelines for the diagnosis of AMR, first proposed at the 10th Banff Conference on Allograft Pathology and refined by a broad-based multidisciplinary panel. Pancreatic AMR is best identified by a combination of serological and immunohistopathological findings consisting of (i) identification of circulating donor-specific antibodies, and histopathological data including (ii) morphological evidence of microvascular tissue injury and (iii) C4d staining in interacinar capillaries. Acute AMR is diagnosed conclusively if these three elements are present, whereas a diagnosis of suspicious for AMR is rendered if only two elements are identified. The identification of only one diagnostic element is not sufficient for the diagnosis of AMR but should prompt heightened clinical vigilance. AMR and ACMR may coexist, and should be recognized and graded independently. This proposal is based on our current knowledge of the pathogenesis of pancreas rejection and currently available tools for diagnosis. A systematized clinicopathological approach to AMR is essential for the development and assessment of much needed therapeutic interventions.

Proteases in Plasma and Kidney of db/db Mice as Markers of Diabetes-Induced Nephropathy.

Db/db mice are overweight, dyslipidemic and develop diabetic complications, relevant for similar complications in human type 2 diabetes. We have used db/db and db/+ control mice to investigate alterations in proteinase expression and activity in circulation and kidneys by SDS-PAGE zymography, electron microscopy, immunohistochemistry, Western blotting, and in situ zymography. Plasma from db/db mice contained larger amounts of serine proteinases compared to db/+ mice. Kidneys from the db/db mice had a significantly larger glomerular surface area and somewhat thicker glomerular basement membranes compared to the db/+ mice. Furthermore, kidney extracts from db/+ mice contained metalloproteinases with M(r) of approximately 92000, compatible with MMP-9, not observed in db/db mice. These results indicate that higher levels of serine proteinases in plasma may serve as potential markers for kidney changes in db/db mice, whereas a decrease in MMP-9 in the kidney may be related to the glomerular changes.

Gene expression and distribution of key bone turnover markers in the callus of estrogen-deficient, vitamin D-depleted rats.

An experimental rat model was used to test the hypothesis that in osteoporosis (OP) the molecular composition of the extracellular matrix in the fracture callus is disturbed. OP was induced at 10 weeks of age by ovariectomy and a vitamin D(3)-deficient diet, and sham-operated animals fed normal diet served as controls. Three months later a closed tibial fracture was made and stabilized with an intramedullary nail. After 3 and 6 weeks of healing, the animals were killed and the fracture calluses examined with global gene expression, in situ mRNA expression, and ultrastructural protein distribution of four bone turnover markers: osteopontin, bone sialoprotein, tartrate-resistant acid phosphatase, and cathepsin K. Global gene expression showed a relatively small number of differently regulated genes, mostly upregulated and at 3 weeks. The four chosen markers were not differently regulated, and only minor differences in the in situ mRNA expression and ultrastructural protein distribution were detected. Gene expression and composition of fracture calluses are not generally disturbed in experimental OP.

Bone marrow mesenchymal stem cells in a hyaluronan scaffold for treatment of an osteochondral defect in a rabbit model.

The purpose of this study was to evaluate the efficiency of using mesenchymal stem cells (MSC) in a hyaluronan scaffold for repair of an osteochondral defect in rabbit knee. Bone marrow was harvested from the posterior iliac crest in 11 New Zealand White rabbits. MSC were isolated and cultured in autologous serum for 28 days and transferred to a hyaluronan scaffold 24 h prior to implantation. A 4 mm diameter and 1.5 mm deep defect was created in the medial femoral condyle of both knees and the scaffold with MSC was implanted in one knee while an empty scaffold was implanted in the contra-lateral knee. After 24 weeks the rabbits were killed and histological sections were subjected to semiquantitative and quantitative evaluation by observers blinded regarding treatment modality. High degree of filling was obtained, but there was no statistically significant difference between the two treatments. However, there was a tendency for a better quality of repair in the MSC treated knees. No hypertrophy was observed by either method. MSC in a hyaluronan scaffold may be a promising treatment approach, but further studies are needed to determine the best combination of scaffold and cells.

Banff schema for grading pancreas allograft rejection: working proposal by a multi-disciplinary international consensus panel.

Accurate diagnosis and grading of rejection and other pathological processes are of paramount importance to guide therapeutic interventions in patients with pancreas allograft dysfunction. A multi-disciplinary panel of pathologists, surgeons and nephrologists was convened for the purpose of developing a consensus document delineating the histopathological features for diagnosis and grading of rejection in pancreas transplant biopsies. Based on the available published data and the collective experience, criteria for the diagnosis of acute cell-mediated allograft rejection (ACMR) were established. Three severity grades (I/mild, II/moderate and III/severe) were defined based on lesions known to be more or less responsive to treatment and associated with better- or worse-graft outcomes, respectively. The features of chronic rejection/graft sclerosis were reassessed, and three histological stages were established. Tentative criteria for the diagnosis of antibody-mediated rejection were also characterized, in anticipation of future studies that ought to provide more information on this process. Criteria for needle core biopsy adequacy and guidelines for pathology reporting were also defined. The availability of a simple, reproducible, clinically relevant and internationally accepted schema for grading rejection should improve the level of diagnostic accuracy and facilitate communication between all parties involved in the care of pancreas transplant recipients.

Screw augmentation in displaced femoral neck fractures. Clinical and histological results using a new composite.

We wanted to assess a new technique for augmentation of parallel screws in internal fixation of displaced femoral neck fractures with a bis-GMA-based composite delivered around the screw head. Twenty-one consecutive patients admitted with displaced femoral neck fractures were operated with internal fixation with two parallel Olmed screws augmented with the composite, and followed for 24 months. The composite was introduced through the lumen of the cannulated screws and deposited in the femoral head around the threaded part of the screws. The procedure of augmenting was technically feasible and operation time was on average 33 min. Eleven patients were re-operated due to healing complications within 24 months. There were five redisplacements, four non-unions and two cases of avascular necrosis. Histological examination of four extracted femoral heads showed fragmentation of the composite into small particles with foreign-body response with giant cells and macrophages along with granulation tissue formation and low grade inflammation. The method of augmentation was technically easy, but the failure rate was high and the fragmentation of the composite with inflammatory response found on histology is noteworthy.

Skin biopsy findings and results of neuropsychological testing in the first confirmed cases of CADASIL in Norway.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is characterized by mutations in the notch3 gene. The mutation can be demonstrated by a gene test. The first group of Norwegian patients with CADASIL confirmed by gene testing was recently described. The present study includes six of the original nine patients with demonstrated notch3 mutation plus one patient with symptoms, who had been unwilling to go through gene testing. These seven patients underwent skin biopsy for electron microscopy. One patient was cognitively too impaired, but six went through neuropsychological testing. By electron microscopy characteristic granular osmiophilic material (GOM) was detected in all skin biopsies. The material is seen as granular deposits in the basal lamina of the smooth muscle layer, often making impressions upon adjacent smooth muscle cells. It seems important that blood vessels over a certain size (diameter >20 microm) are examined. A varying extent of cognitive decline was noted amongst all by neuropsychological testing. In cases with multiple infarctions reduction of perceptual speed, visuospatial skills and working memory could be demonstrated. Four patients showed executive dysfunction.

Surgical treatment of osteochondroma of the mandibular condyle in the adult. A 5-year follow-up.

Five adult patients with temporomandibular joint (TMJ) pain and impaired mandibular function and with clinical and radiographic features of unilateral osteochondroma of the mandibular condyle was included in a 5-year prospective follow-up study. All patients were surgically treated with condylectomy and reshaping of the condylar neck which was then positioned underneath the preserved TMJ disk. The yearly follow-up evaluations comprised measurements of maximum interincisal opening and protrusive movements, assessments of occlusion and TMJ pain as well as tomographic interpretation of recurrent growth. No patient showed recurrence of growth at the 5-year follow-up and mandibular function and occlusion was normalized in all patients. The results indicate that this conservative surgical approach can be recommended for treatment of osteochondroma of the mandibular condyle.

Vascularisation and osteochondrosis of the epiphyseal growth cartilage of the distal femur in pigs--development with age, growth rate, weight and joint shape.

Until recently, the cartilage canals of the epiphyseal growth cartilage have not been associated with any specific disease. However, data support the hypothesis that osteochondrosis could be related to inadequate blood supply from vessels in these channels. We have done a study to investigate the relationship between the regression of cartilage canals and the formation of osteochondrosis latens in the epiphyseal growth cartilage of the distal femur in pigs, and the relationship between these events and age, growth rate, weight and femoral shape of the individual animals. This involved, in part, a comprehensive study of the distribution and pattern of regression of the cartilage canals. We found that the regression is a highly predictable process that follows an age-dependent pattern. However, we failed to prove any association between overall vascular regression and osteochondrosis, between vascular regression and weight, growth rate or femoral shape or between osteochondrosis and weight, growth rate or femoral shape. This may indicate that osteochondrosis latens is not caused by a general failure of vascular supply or general factors such as growth rate, but rather a consequence of local conditions affecting a limited number of vessels. A factor fitting this description is local compression.

Inflammatory cell and cytokine patterns in patients with painful clicking and osteoarthritis in the temporomandibular joint.

The occurrence of a subset of cytokines and leukocytes in the posterior disc attachment area of the temporomandibular joint (TMJ) was investigated in two patient groups, i.e, one group with painful clicking and one with osteoarthritis. Synovial biopsies were taken during discectomy in 19 patients with painful clicking and 20 with osteoarthritis. One set of specimens was examined with immunohistochemistry, using frozen sections postfixed by para-formaldehyde and with the cell membranes permeablized in saponin. These sections were incubated with antibodies against cytokines IL-1alpha, IL-1beta, IL-1ra, TNFalpha, IFNgamma, IL2 in all patients and TGFbeta1,2,3 in 16. The other set of specimens was used to characterize cell infiltrates using immunohistochemistry with monoclonal antibodies against antigens CD68 and CD45RO, respectively. Moreover, PCNA was included as a marker for cell proliferation. The cytokine staining was most frequently positive for IL-1alpha and IL-1beta in both patient groups. However, joints with OA showed a more complex cytokine pattern, also involving IFN-gamma (P = 0.019), IL-ra (P = 0.047), and apparently but without reaching the chosen level of significance, IL-2, TNF-alpha and TGF-beta1,2,3. Positive staining for CD45RO was frequent in both groups. OA patients showed more frequently positive staining for CD68 (P = 0.025) and apparently for PCNA.

Synovial chondromatosis of the temporomandibular joint: clinical, surgical and histological aspects.

Nine patients with histologically confirmed unilateral synovial chondromatosis of the temporomandibular joint were treated surgically with extirpation of loose bodies and partial synovectomy. In six of them the histological material was available for a systematic examination. The results of treatment were evaluated clinically and with MRI after a follow-up ranging between 1 and 17 years. Our findings suggest that synovial chondromatosis of the temporomandibular affects only the synovial lining of the upper compartment. The histological appearance is that of a benign chronic inflammation varying in severity and with metaplastic activity. The most specific clinical sign of synovial chondromatosis is swelling over the joint. Distension of the lateral capsule and fluid in the joint on the MRI are very suggestive of this diagnosis. Loose bodies also indicate synovial chondromatosis, but they are not always detected on the preoperative MRI. The surgical treatment should be conservative and include thorough removal of the loose bodies and partial synovectomy in areas with marked inflammation.

Recurrent angiolymphoid hyperplasia with eosinophilia mimicking temporal arteritis associated with nephrotic syndrome.

We report on a middle-aged Caucasian male who presented with nephrotic syndrome that on 2 consecutive recurrences was accompanied by a pulsating tumor suggesting temporal arteritis. Renal biopsies showed features of a low-grade mesangial-proliferative glomerulonephritis. The resected tumor in the temporal region revealed a lesion consistent with angiolymphoid hyperplasia with eosinophilia (ALHE), with moderate inflammatory involvement of the temporal artery. The patient was successfully treated with oral prednisolone in addition to removal of the tumor, but has remained steroid-dependent. To our knowledge, only 2 cases of ALHE and nephrotic syndrome have been reported so far in non-Japanese individuals [Altman et al. 1995, Sonkodi et al. 1987], and we are not aware of any previous case combining these features while simultaneuosly mimicking temporal arteritis.

Ultrastructural distribution of osteoadherin in rat bone shows a pattern similar to that of bone sialoprotein.

Osteoadherin (OSAD) is a keratan sulfate proteoglycan recently isolated from bovine and rat bone. Based on results obtained from in vitro experiments, the protein was shown to bind osteoblasts via the integrin receptor alpha v beta 3. Due to OSAD's capacity to bind hydroxyapatite crystals, a role for the protein in the mineralization process has also been suggested. To test these hypotheses in an in vivo model, the ultrastructural localization of OSAD in bone, tibial (metaphyses and diaphyses), and calvarial samples from normal 10 to 12-day-old rats were examined by immunohistochemical techniques at the ultrastructural level. In addition to the qualitative studies, quantitative measurements of OSAD marker density were performed in relevant compartments. Immunolabeling for OSAD was located to the mineralized bone matrix, with highest concentration of marker at the border between bone and cartilage remnants in the metaphyseal trabeculi. Intracellular labeling was low and no systemic accumulation of OSAD markers was observed at the cell-matrix interface. The observed distribution pattern of OSAD is strikingly similar to that of bone sialoprotein (BSP), confirmed by double labeling. The results of the current study support a role for OSAD in the mineralization process. In this process BSP is assumed to be a nucleator of hydroxyapatite crystals, and OSAD could work in concert with BSP to regulate nucleation. However, the mechanisms involved remain to be elucidated.

Fibromodulin-null murine knee joints display increased incidences of osteoarthritis and alterations in tissue biochemistry.

OBJECTIVE: To elucidate variations in tissue ultrastructure and incidence of pathology between fibromodulin (FM)-null mice and wild-type (WT) animals. DESIGN: FM-null and WT siblings from different age groups were compared. Serial sections were made through paraffin-embedded whole knees and investigated histologically. Additionally, medial femoral condyle peaks from sibling pairs were investigated ultrastructurally using transmission electron microscopy. RESULTS: Histological findings demonstrated a clear and increasing disparity between tissue degeneration in WT and FM-null animal knees with progressing age. Distinct differences were apparent by 36 weeks. Around the 80 week period and onward these differences became profound. However, qualitative ultrastructural investigation did not indicate either any aberrant tissue ultrastructure or any abnormal collagen fibril forms in FM-null articular cartilage compared with WT. Biochemical and immunohistochemical investigation of FM-null articular cartilage showed a significant increase in tissue levels of lumican (LUM). Conversely, the cruciate ligaments of the knee showed both an increase in LUM content and considerable structural abnormalities including the tendency towards rupture. CONCLUSION: This report indicates for the first time that FM-null mice have a higher propensity towards degenerative changes in their knee joints than comparable WT animals. Interestingly, no underlying ultrastructural or fibril abnormalities within the articular cartilage could be identified to explain why FM-null cartilage is more prone to pathological changes than wild-type tissue. We conclude that alterations in ligaments, and possibly other tissues within the knee, are of considerable importance in the pathogenesis of the observed articular cartilage degeneration.