Osteocyte differentiation and the formation of an interconnected cellular network in vitro.

Extracellular matrix (ECM) stiffness and cell density can regulate osteoblast differentiation in two dimensional environments. However, it is not yet known how osteoblast-osteocyte differentiation is regulated within a 3D ECM environment, akin to that existing in vivo. In this study we test the hypothesis that osteocyte differentiation is regulated by a 3D cell environment, ECM stiffness and cell density. We encapsulated MC3T3-E1 pre-osteoblastic cells at varied cell densities (0.25, 1 and 2 × 106 cells/mL) within microbial transglutaminase (mtgase) gelatin hydrogels of low (0.58 kPa) and high (1.47 kPa) matrix stiffnesses. Cellular morphology was characterised from phalloidin-FITC and 4',6-diamidino-2-phenylindole (DAPI) dilactate staining. In particular, the expression of cell dendrites, which are phenotypic of osteocyte differentiation, were identified. Immunofluorescent staining for the osteocytes specific protein DMP-1 was conducted. Biochemical analyses were performed to determine cell number, alkaline phosphatase activity and mineralisation at 2.5 hours, 3, 21 and 56 days. We found that osteocyte differentiation and the formation of an interconnected network between dendritic cells was significantly increased within low stiffness 3D matrices, compared to cells within high stiffness matrices, at high cell densities. Moreover we saw that this network was interconnected, expressed DMP-1 and also connected with osteoblast-like cells at the matrix surface. This study shows for the first time the role of the 3D physical nature of the ECM and cell density for regulating osteocyte differentiation and the formation of the osteocyte network in vitro. Future studies could apply this method to develop 3D tissue engineered constructs with an osteocyte network in place.

Cell morphology and focal adhesion location alters internal cell stress.

Extracellular mechanical cues have been shown to have a profound effect on osteogenic cell behaviour. However, it is not known precisely how these cues alter intracellular mechanics to initiate changes in cell behaviour. In this study, a combination of in vitro culture of MC3T3-E1 cells and finite-element modelling was used to investigate the effects of passive differences in substrate stiffness on intracellular mechanics. Cells on collagen-based substrates were classified based on the presence of cell processes and the dimensions of various cellular features were quantified. Focal adhesion (FA) density was quantified from immunohistochemical staining, while cell and substrate stiffnesses were measured using a live-cell atomic force microscope. Computational models of cell morphologies were developed using an applied contraction of the cell body to simulate active cell contraction. The results showed that FA density is directly related to cell morphology, while the effect of substrate stiffness on internal cell tension was modulated by both cell morphology and FA density, as investigated by varying the number of adhesion sites present in each morphological model. We propose that the cells desire to achieve a homeostatic stress state may play a role in osteogenic cell differentiation in response to extracellular mechanical cues.

[Histological lesions of supraspinatus tendons in full thickness tears of the rotator cuff]. / Les lésions histologiques des tendons des supraspinatus dans les ruptures transfixiantes de la coiffe des rotateurs.

PURPOSE OF THE STUDY: Iterative tears after simple tendon bone suture of transfixiant ruptures of the rotator cuff are frequent. The frequency of iterative ruptures had seemed to be correlated to the importance of the preoperative fatty degenerations of the cuff muscles. But fatty degeneration cannot account for iterative ruptures occurring with no or little preoperative muscle fatty degeneration. The authors have tried to know if iterative ruptures could also be accounted for by the presence on repaired tendinous stumps of histological lesions which are known to lower their mechanical quality, and if such was the case, they have tried to know what could be the impact of these lesions on the repairing technique by suturing cuff ruptures. MATERIAL AND METHOD: Thirty-two distal stumps of ruptured supraspinatus, resected on more than 1 cm to eliminate macroscopic tendinous lesions (thin, lacerated, cleaved, hardened or whitish tendons) and to reach a new stump, slightly bleeding and thick enough to hold the stitches correctly, have been studied on longitudinal histological slides. RESULTS: All the tendinous resected stumps had histological anomalies: dilacerations lesions of the conjunctive tissue, fatty infiltrations, necrosis, scar lesions and micro calcifications were found. The histological lesions were often associated. The histological lesions were an average 4 mm shorter than those of the tendinous resection. In 18 cases however the whole of the resected tendon was histologically abnormal. DISCUSSION: Tendinous histological lesions found on the whole or almost the whole length of the macroscopically abnormal tendinous stumps diminish the mechanical quality of the tendons. Insufficient resection of the tendinous stumps leaving macroscopical lesions and therefore histological lesions could account for iterative ruptures in the first post operative year. Yet complete resection of the macroscopically abnormal tendinous stump does not entail that the neo tendinous stump to be repaired is histologically normal. This encourages to put the stitches well within the zone of the tendinous resection and to bury the tendinous stumps into a highly vascular bony trough. But tendinous resection although logical from the histological and mechanical point of view widens the rupture of the cuff to be repaired. It can make sutures under tension, which can entail iterative ruptures. Musculo-tendinoplasty appears then necessary to perform sutures without tension. CONCLUSION: Histological lesions of tendinous stumps of cuff ruptures correspond roughly to tendinous macroscopic lesions. They could be at the origin of iterative ruptures that take place after simple tendon-bone sutures of ruptured cuffs which are not accounted for by muscular fatty degeneration. They could also partly account for iterative ruptures taking place when pre operative fatty degeneration is evident. It seems logical to resect macroscopically abnormal tendinous stumps before performing a suture. But the tendon-bone suture without tension is then often possible only with musculo-tendinoplasties.

Hemarthrosis: an unusual complication of type 1 neurofibromatosis.

Type 1 neurofibromatosis is one of the most common autosomal-dominant disorders and often includes orthopedic manifestations. We report the case of a 48-year-old woman with hemarthrosis caused by a popliteal cyst infiltrated by a diffuse neurofibroma associated with angiodysplasia. Surgical resection of this tumor was followed by postoperative hemorrhagic complications.

Macroscopic and histological assessments of the cruciate ligaments in arthrosis of the knee.

We examined the macroscopic appearance of both cruciate ligaments in 52 knees during knee replacement. It was classsified as normal, abnormal or ruptured. The ligaments were also evaluated histologically: stage 0 (normal), stage I (degeneration of < 1/3 of the collagen fibers), stage 2 (degeneration of 1/3-2/3) and stage 3 (> 2/3). 17 anterior cruciate ligaments (ACL) were normal, 14 were abnormal and 21 ruptured. All the posterior cruciate ligaments (PCL) were normal. 14 ACL were stage 0, 6 stage 1, 8 stage 2 and 24 were stage 3. 22 PCL were stage 0, 14 stage 1, 13 stage 2 and 3 were stage 3. When the ACL was abnormal or ruptured, the PCL was stage 0 only in one fourth of the cases. The long-term results of TKR retaining the PCL should be better if the ligament is strong. The intraoperative assessment of the macroscopic appearance of the ACL reflects the histological state of the PCL.

Malignant peripheral nerve sheath tumors associated with neurofibromatosis type 1: a clinicopathologic and molecular study of 17 patients.

OBJECTIVE: To identify potential prognostic factors and criteria for early detection of malignant peripheral nerve sheath tumors associated with neurofibromatosis type 1 (NF1). DESIGN: Retrospective study of malignant peripheral nerve sheath tumors in a cohort of 395 patients with NF1 followed up between October 1, 1988, and January 1, 1999; review of the clinical and histological characteristics of treatment and course; and analysis of p53 mutations and overexpression in tumors. SETTING: Teaching hospital referral neurofibromatosis center for adults. PATIENTS: Seventeen patients with NF1 (9 males and 8 females). Mean +/- SD patient age at diagnosis was 32 +/- 14 years. MAIN OUTCOME MEASURES: (1) Clinical symptoms, (2) comparison of p53 mutations and overexpression in benign vs malignant tumors; and (3) median survival. RESULTS: Twelve patients had high-grade tumors. All tumors except 1 developed on preexisting nodular or plexiform neurofibromas. Pain and enlarging mass were the first and predominant signs. None of the benign tumors displayed significant p53 staining or p53 mutations. Six of 12 malignant tumors significantly overexpressed p53, and 4 of 6 harbored p53 missense mutations. Median survival was 18 months overall, 53 months in peripheral locations, and 21 months in axial locations. CONCLUSIONS: Malignant peripheral nerve sheath tumors are highly aggressive in NF1. They mostly arise from plexiform or nodular neurofibromas. Investigations and deep biopsy of painful and enlarging nodular or plexiform neurofibromas should be considered in patients with NF1. Late appearance of p53 mutations and overexpression precludes their use as predictive markers of malignant transformation.

Iliac vein thrombosis complicating SAPHO syndrome: MRI and histologic features of soft tissue lesions.

Subclavian and superior vena cava obstruction complicating SAPHO (Synovitis, Acne, Pustulosis, Hyperostosis, Osteitis) syndrome has been described. We report the first case to our knowledge of iliac vein thrombosis complicating lumbar vertebral osteitis due to SAPHO syndrome. Lumbar MRI demonstrated a large tissue mass anterior to the involved vertebras and surrounding the right iliac vein. Histology of the mass showed aseptic inflammation.

Immunohistological study of entheses in spondyloarthropathies: comparison in rheumatoid arthritis and osteoarthritis.

OBJECTIVE: To determine which inflammatory cell types are present in entheses from patients with spondyloarthropathy (SpA) compared with patients with rheumatoid arthritis (RA) or osteoarthritis (OA). METHODS: Enthesis specimens were obtained during orthopaedic procedures in eight patients with SpA, four with RA, and three with OA. After decalcification, the lymphocyte subsets (CD3, CD4, CD8, CD20) in the bone marrow component of each enthesis were measured by an immunohistochemical technique. RESULTS: Oedema and an inflammatory infiltrate were present in all the SpA specimens, being clearly predominant in the bone marrow component of the entheses. The density of all cell types in the bone marrow was significantly higher in the SpA group than in the two other groups. The cell type CD3+ showed the greatest difference between the SpA and RA groups, being increased fivefold in the SpA group. Within the SpA group, CD3+ cells were considerably more numerous than CD20+ cells-a difference from the RA group-and the predominant T cells were CD8+. CONCLUSION: Persistent oedema with an inflammatory infiltrate composed predominantly of CD8+ cells was noted in the entheses of patients with SpA, being predominant in the bone marrow. These results suggest that CD8+ cells may have a key role in local inflammation in SpAs.

Radiation-induced bone sarcoma following total body irradiation: role of additional radiation on localized areas.

A 44-year-old patient who had had acute monoblastic leukemia developed an osteosarcoma of the pelvic bones 5 years after an allogeneic bone marrow transplant from his HLA-identical sister. He had additionally received superficial cutaneous radiation of the legs and pelvis, over the 3 weeks prior to total body irradiation (TBI), because of cutaneous leukemic lesions. The tumor was a fibrohistiocytomatous osteogenic sarcoma. The first lesion was in the right ilium, and a second lesion appeared 18 months later, symmetrically on the left ilium. Despite treatment, the patient died from metastases. At the time of diagnosis of radiation-induced sarcoma, the patient was free of leukemia and had several risk factors already reported to favor the development of solid tumors in stem cell recipients. These include acute leukemia, TBI and graft-versus-host disease. As he developed symmetrical lesions of the pelvic bone, and because of the histology of the radiation-induced tumor, we assumed that the additional radiation of the skin prior to TBI may have contributed to the pathogenesis of this malignant fibrous histiocytoma. Therefore, the risk/benefit ratio should be carefully considered in unusual indications. These patients should benefit from a close follow-up of the superimposed areas.

Poor eight-year survival of cemented zirconia-polyethylene total hip replacements.

Between January 1988 and January 1991 we performed 100 consecutive cemented total hip replacements using a zirconia head, a titanium alloy stem and a polyethylene cup. We reviewed 78 of these hips in 61 patients in detail at a mean of 5.8 years (1 to 9). Aseptic loosening was seen in 11 hips (14%). Eight needed revision. In total, 37 cups (47.5%) showed radiolucent lines, all at the cement-bone interface, with 18 (23%) involving all the interface. Of the 78 femoral implants, 17 (21.7%) showed radiolucent lines, and two, which had a complete line of more than 1 mm thick, definite endocortical osteolyses. There was also an abnormally high incidence of osteolysis of more than 2 mm at the calcar. Survivorship analysis showed that only 63% were in situ at eight years. These worrying results led us to abandon the use of zirconia heads, since at the same hospital, using the same femoral stem, cement and polyethylene cup, but with alumina femoral heads, the survival rate was 93% at nine years. We discuss the possible reasons for the poor performance of zirconia ceramic.

Labial salivary gland biopsy for diagnosis of amyloid polyneuropathy.

Biopsies of peroneal nerve and labial salivary gland (LSG) were performed in 32 patients with polyneuropathy of unknown origin. Amyloid deposits were detected in 7 LSG (transthyretin=5; amyloid, light chain derived=2) and 6 nerve biopsies. Familial amyloid and light chain amyloid polyneuropathies were subsequently confirmed by relevant tests. We propose that LSG biopsy, a minimally invasive test that may document both sicca syndrome and amyloidosis, should be systematically performed in the investigation of patients with axonal polyneuropathies.

[Pigmented nodular tenosynovitis. A misdiagnosed etiology of isolated tumefaction of fingers or toes]. / Ténosynovite nodulaire hémopigmentée. Une étiologie méconnue de tuméfaction isolée des doigts et des orteils.

BACKGROUND: Pigmented nodular tenosynovitis (more commonly called giant cell tumor) is a benign tumor of synovial joint or tendon sheaths. CASE REPORT: A 17-year-old girl had a tumefaction of the first toe. The mass was firm and painless and had been present for one year. Radiological studies revealed a cystic area of the first phalanx. Magnetic resonance imaging demonstrated a poorly vascularized tissular lesion. Surgical excision of the tumor was performed. Macroscopic and histologic findings confirmed the diagnosis of pigmented nodular tenosynovitis. DISCUSSION: Clinical and radiological aspects of nodular tenosynovitis are characteristic. This diagnosis should be made by dermatologists. Eighty percent of cases occur in fingers and more rarely in toes. Patients are usually females (60 p. 100). A subcutaneous mass is the most common presenting sign whereas pain or joint swelling are rare. Optimal treatment is surgical resection. The only risk is recurrence (20 p. 100).

Unusual CD3+, CD4+ large granular lymphocyte expansion associated with a solid tumor.

Large granular lymphocyte proliferation has been reported in association with rheumatoid arthritis. We report an unusual association between a pleural mesothelioma and an unusual form of large granular proliferation of CD3+, CD4+, CD8-, CD56+ cells. This case sheds light on the possible causal relationship between these 3 conditions.

Prognostic factors in patients with localized Ewing's sarcoma: the effect on survival of actual received drug dose intensity and of histologic response to induction therapy.

To bring to the fore the most important prognostic factors in Ewing's sarcoma (ES) with current protocols, we studied the classical prognostic factors, dose intensity (DI) of actual received drugs, age and histological response to induction therapy and their correlation in 39 patients with localized ES treated from 11/85 to 06/95 to identify eventual predictors of event-free survival (EFS). Inclusion criteria were age 35 yr or less, definitive local treatment by our team and chemotherapy including at least 4 drugs: vincristine (VCR), dactinomycin (DACT), doxorubicin (DOXO) cyclophosphamide (CPX). The endpoint was the absence of relapse. Parameters related to the status of patients were tested using the Chi square test or Fisher's exact test. The non parametric Kruskal-Wallis test was used for quantitative data. When necessary stratified analysis was done using the Mantel Cox test. With a median follow-up of 7 yr, overall survival (OS) and EFS were both 67% at 7 yr. According to univariate analysis, the significant predictors of survival were the DI of VCR and DACT, the histological response to preoperative chemotherapy (CT), the patient's age (< 18 yr DFS: 84%; > 18 yr DFS: 38%). The risk of metastases was almost tenfold higher in patients with low received DI of VCR (DFS 40% versus 95%) and of DACT (DFS 48% versus 94%). The prognostic value of primary tumor characteristics (tumoral volume or location) was erased by the comprehensive treatment. Following multivariate analysis, the actual received DI of VCR (p < 0.02) and DACT (p < 0.03) and the histological response to preoperative CT (p < 0.05) were retained as the only significant independent predictors of EFS. Taking into account the actual received DI of VCR and DACT, the prognostic value of age disappears. In conclusion, this study points out the main role of the drug DI in ES (particularly VCR and DACT) and of histological response to preoperative CT.

[Additive medial osteotomy of the tibia locked with a coral callus. First results apropos of 38 operations]. / Ostéotomie tibiale d'addition interne calée par un coin corail. Premiers résultats à propos de 38 interventions.

We inserted a calibrated coral callus to lock addive medial osteotomy of the tibia in 38 patients. We followed these patients for 1 to 6 years evaluating clinical, radiological and histological outcome. Rehabilitation was very insufficient. In our later cases, the porosity of the coral insert was improved, apparently facilitating bone penetration.

[Primary hyperparathyroidism. Optimization of surgical results with systematic preoperative 99mTc-Sestamibi scintigraphy]. / Hyperparathyroïdie primaire. Optimisation des résultats chirurgicaux par la scintigraphie au 99mTc-Sestamibi pré-opératoire systématique.

OBJECTIVE: The importance of preoperative 99mTc Sestamibi (MIBI) scintigraphy in case of reoperation for persistent hyperparathyroidism is well recognized, but it use as a systematic exploration technique remains a question of debate. We conducted this study to determine whether preoperative MIBI scans performed in all cases before surgery have any real impact. METHOD: Two successive series of 65 operated patients were included in the study. In the first group, the MIBI scan was not performed prior to surgery while in the second group the MIBI scan was part of the systematic work-up. RESULTS: The sensitivity and positive predictive value of MIBI were 92% and 96% respectively. Sensitivity for unique adenomas was 95% and 80% for multiple forms. In the first group without systematic scans, there were two unproductive procedures. In the second group, all procedures were productive and no reoperations were required. Two mediastinal adenomas were removed at the first cervicotomy in this group. The rate of complications was similar for both groups. Mean operation time was 2 hours in the first group and 1 hour 30 minutes for the second. CONCLUSION: Our series shows that there are three main advantages of using MIBI scintigraphy systematically prior to surgery for hyperparathyroidism: the procedure is easier in patients with cervical adenomas, particularly in case of ectopic localizations; the mean duration of the operation is shortened by 30 minutes; mediastinal ectopic localizations can be removed by sternotomy at the first operation. These advantages appear to be great enough to propose systematic use of MIBI scan prior to surgery. When MIBI scan shows a single gland, the risk of missing a multiple localization is less than 2% in our experience. It appears possible to operate under local anesthesia via a single-sided approach in elderly patients or high-risk patients.

[Solitary infantile myofibromatosis of bone]. / Myofibromatose infantile osseuse isolée.

We report one case of Solitary infantile Myofibromatosis of Bone in a 14-year old girl. Radiologic features of the tibial lesion were consistent with the diagnosis of a benign bone tumor. The final diagnosis was made on a total resection of the tumor. Histologically, the tumor consisted of nodules, hyalinized or cellular, with spindle-shaped cells resembling fibroblasts or smooth-muscle cells. There were numerous vascular spaces in close contact with these nodules. The tumor cells were immunoreactive with anti-alpha-smooth muscle actin antibodies. These data were consistent with the diagnosis of Myofibromatosis. The histologic features were similar to those of Infantile Myofibromatosis of other sites such as skin and soft tissue. The case reported is rare and not yet published, due to the age and sex of the patient, and the location of the tumor.