Age and synovitis affect the results of the treatment of knee osteoarthritis with Microfragmented Autologous Fat Tissue.

PURPOSE: This study aims to assess the effectiveness of Microfragmented Autologous Fat Tissue (MFAT) treatment for knee osteoarthritis and to investigate whether patients' pre-treatment clinical condition, such as synovitis, correlates with clinical outcomes, to identify potential predicting factors for the success or failure of the treatment. METHODS: In this prospective Cohort Study Level II multicentric trial, consecutive patients with a diagnosis of early/mild osteoarthritis and failure of previous conservative measures were enrolled to undergo diagnostic arthroscopy and a single MFAT injection. Patients were assessed with repeated scoring systems at baseline, 6 months, and 12 months after surgery. The demographic features, the arthroscopic findings, the immunophenotype of injected tissue and the histologic examination of synovia of failed patients were analyzed. RESULTS: Data from 91 patients showed a significant improvement in Lysholm, WOMAC scores at 1-year follow-up (p < 0.001). A significant decrease in VAS score was observed, while a significant improvement of measured flexion angle was registered at 1 year (p < 0.001). No major complications were reported. Age and synovitis were identified as significant factors influencing the clinical outcome (p < 0.05). Body mass index, previous or concomitant procedures, and specific cartilage defects had no influence. The mean number of injected adipose tissue-derived mesenchymal stem cells seem not to correlate with the clinical outcome. CONCLUSION: MFAT is effective in reducing pain when used with a single dose injection in early/mild OA of the knee, without major complications. Age over 60 and synovitis may be predictive for persistent pain at one year and should be considered before indications.

Primary breast cancer stem-like cells metastasise to bone, switch phenotype and acquire a bone tropism signature.

BACKGROUND: Bone metastases represent a common and severe complication in breast cancer, and the involvement of cancer stem cells (CSCs) in the promotion of bone metastasis is currently under discussion. Here, we used a human-in-mice model to study bone metastasis formation due to primary breast CSCs-like colonisation. METHODS: Primary CD44⁺CD24⁻ breast CSCs-like were transduced by a luciferase-lentiviral vector and injected through subcutaneous and intracardiac (IC) routes in non-obese/severe-combined immunodeficient (NOD/SCID) mice carrying subcutaneous human bone implants. The CSCs-like localisation was monitored by in vivo luciferase imaging. Bone metastatic CSCs-like were analysed through immunohistochemistry and flow cytometry, and gene expression analyses were performed by microarray techniques. RESULTS: Breast CSCs-like colonised the human-implanted bone, resulting in bone remodelling. Bone metastatic lesions were histologically apparent by tumour cell expression of epithelial markers and vimentin. The bone-isolated CSCs-like were CD44⁻CD24⁺ and showed tumorigenic abilities after injection in secondary mice. CD44⁻CD24⁺ CSCs-like displayed a distinct bone tropism signature that was enriched in genes that discriminate bone metastases of breast cancer from metastases at other organs. CONCLUSION: Breast CSCs-like promote bone metastasis and display a CSCs-like bone tropism signature. This signature has clinical prognostic relevance, because it efficiently discriminates osteotropic breast cancers from tumour metastases at other sites.

Teriparatide increases the maturation of circulating osteoblast precursors.

UNLABELLED: This study shows that teriparatide promotes the circulating osteoblast (OB) precursor degree of maturation in patients affected by postmenopausal osteoporosis. INTRODUCTION: Anabolic treatment with teriparatide has proven effective for the therapy of postmenopausal osteoporosis and significantly reduces the risk of non-vertebral fragility fractures. The aim of this study was to investigate the effect of teriparatide on circulating OB precursors. METHODS: We evaluated by flow cytometry and real-time PCR the expression of OBs typical markers in peripheral blood mononuclear cells during treatment with teriparatide plus calcium and vitamin D, raloxifene plus calcium and vitamin D or calcium and vitamin D alone at various time points. Serum bone alkaline phosphatase and osteocalcin (OC) were measured as markers of bone turnover. RESULTS: Our results show that circulating OB precursors are more numerous and more immature in patients affected by fragility fractures than in osteoporotic patients without fractures. We also show that teriparatide treatment increases the expression of alkaline phosphatase and of OC in OB precursors; thus, it increases their degree of maturation. CONCLUSIONS: We suggest that teriparatide acts as anabolic agents also by promoting the maturation of OB precursors.

Bone and bone marrow pro-osteoclastogenic cytokines are up-regulated in osteoporosis fragility fractures.

UNLABELLED: This study evaluates cytokines production in bone and bone marrow of patients with an osteoporotic fracture or with osteoarthritis by real time PCR, Western blot and immunohistochemistry. We demonstrate that the cytokine pattern is shifted towards osteoclast activation and osteoblast inhibition in patients with osteoporotic fractures. INTRODUCTION: Fragility fractures are the resultant of low bone mass and poor bone architecture typical of osteoporosis. Cytokines involved in the control of bone cell maturation and function are produced by both bone itself and bone marrow cells, but the roles of these two sources in its control and the amounts they produce are not clear. This study compares their production in patients with an osteoporotic fracture and those with osteoarthritis. METHODS: We evaluated 52 femoral heads from women subjected to hip-joint replacement surgery for femoral neck fractures due to low-energy trauma (37), or for osteoarthritis (15). Total RNA was extracted from both bone and bone marrow, and quantitative PCR was used to identify the receptor activator of nuclear factor kB Ligand (RANKL), osteoprotegerin (OPG), macrophage colony stimulating factor (M-CSF), transforming growth factor ß (TGFß), Dickoppf-1 (DKK-1) and sclerostin (SOST) expression. Immunohistochemistry and Western blot were performed in order to quantify and localize in bone and bone marrow the cytokines. RESULTS: We found an increase of RANKL/OPG ratio, M-CSF, SOST and DKK-1 in fractured patients, whereas TGFß was increased in osteoarthritic bone. Bone marrow produced greater amounts of RANKL, M-CSF and TGFß compared to bone, whereas the production of DKK-1 and SOST was higher in bone. CONCLUSIONS: We show that bone marrow cells produced the greater amount of pro-osteoclastogenic cytokines, whereas bone cells produced higher amount of osteoblast inhibitors in patients with fragility fracture, thus the cytokine pattern is shifted towards osteoclast activation and osteoblast inhibition in these patients.

Increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells in phenylketonuria.

Phenylketonuria (PKU) is commonly complicated by a progressive bone impairment of uncertain aetiology. The therapeutic phenylalanine (Phe)-restricted diet and the possible noxious effects of high plasma Phe concentrations on bone have previously been suggested as possible determinant factors. Since osteoclasts are involved in bone reabsorption, they could play a role in determining bone damage in PKU. The reported increased excretion of bone resorption markers in PKU patients is consistent with this hypothesis. Although different diseases characterized by bone loss have been related to increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells (PBMCs), to date there is no evidence of increased osteoclast formation in PKU. In this study, we compared the spontaneous osteoclastogenesis from PBMCs in 20 patients affected by PKU with that observed in age- and sex-matched healthy subjects. Phenylketonuric patients showed the number of osteoclasts to be almost double that observed in controls (159.9 ± 79.5 and 87.8 ± 44.7, respectively; p = 0.001). Moreover, a strict direct correlation between the spontaneous osteoclastogenesis in PKU patients and the mean blood Phe concentrations in the preceding year was observed (r = 0.576; p = 0.010). An imbalance between bone formation and bone resorption might explain, at least in part, the pathogenesis of bone loss in this disease. These findings could provide new insights into the biological mechanisms underlying bone damage in PKU.

Spontaneous osteoclastogenesis is a predictive factor for bone metastases from non-small cell lung cancer.

Lung cancer is a widespread disease and its incidence is growing. Since therapies have increased the life expectancy of lung cancer patients, the development of bone osteolytic metastases is becoming a common cause of morbidity. Osteolysis is caused by an increased osteoclast activity and may be reduced by inhibiting their formation and activity. We studied 60 male patients affected by NSCLC, divided in early and advanced stage disease. Patients' blood and urinary samples were collected at tumor diagnosis and at follow-up. PBMCs were cultured to investigate the spontaneous osteoclastogenesis. IL-7 was dosed in serum and its quantitative gene expression was evaluated on tumor and healthy tissues by RQ-PCR. Both at diagnosis and follow-up, osteolytic bone patients showed high spontaneous osteoclastogenesis level compared to non-bone metastatic and healthy controls. The presence of spontaneous osteoclastogenesis correlated with urinary crosslinks increase. Serum IL-7 levels were higher in bone metastatic patients than in patients without bone lesions and healthy controls. The serum IL-7 increase correlated with the osteoclastogenesis and, at least in part, depended on an increased IL-7 production by tumor cells. At follow-up, patients with increased osteoclastogenesis and serum IL-7 levels, were subjected to standard clinical analysis, which showed early secondary bone lesions. The in vitro assay for spontaneous osteoclastogenesis and serum IL-7 dosage could be useful for diagnostic purposes and it might be able to monitor cancer patients with a high risk to develop osteolytic metastases at follow-up, especially after a curative treatment.

Reversal of multidrug-resistance using Valspodar (PSC 833) and doxorubicin in osteosarcoma.

High-grade osteosarcoma is an extremely aggressive neoplasm, where over 80% of patients present with life-threatening micrometastases at diagnosis. Systemic control of the disease is therefore critical for the treatment of these patients and neoadjuvant chemotherapy using various drugs, including doxorubicin (DXR), which has been demonstrated to be the most effective regimen. Multidrug resistance (MDR) to some anticancer agents, including DXR, mediated by the MDR1 gene product P-glycoprotein (Pgp), has been shown to be a major cause of chemotherapy failure in osteosarcoma. We analyzed the effect of a cyclosporine A derivate Valspodar (PSC 833) on MDR human osteosarcoma cells. We also evaluated Pgp expression in sporadic appendicular canine osteosarcoma. Moreover, dogs were treated with combined administration of DXR and PSC 833. Several blood samples were collected for the determination of DXR and PSC 833 levels. PSC 833 induced a complete reversal of the resistant phenotype at concentrations compatible with the clinical use. Pgp was present in 12/18 (66.6%) of the cases. At the time of DXR administration, adequate blood concentrations of PSC 833, to provide a complete MDR reversal, were obtained without clinical or laboratory findings of toxicity. Combination therapy with DXR and PSC 833 allowed a 30% decrease in DXR dose infusion with equivalent therapeutic exposure. The high incidence of Pgp expression in osteosarcoma confers to the study a rationale for an effective regimen based on down-modulation of MDR.

Role of Paracoccidioides brasiliensis cell wall fraction containing beta-glucan in tumor necrosis factor-alpha production by human monocytes: correlation with fungicidal activity.

The polysaccharide fraction of Paracoccidioides brasiliensis mycelial cell wall (F1 fraction), the active component of which is composed of beta-glucan, was investigated in regard to the activation of human monocytes for fungal killing. The cells were primed with interferon-gamma (IFN-gamma) or F1 (100 and 200 microg ml(-1)) or F1 (100 and 200 microg ml(-1)) plus IFN-gamma for 24 h and then evaluated for H2O2 release. In other experiments, the cells were pretreated with the same stimuli, challenged with a virulent strain of P. brasiliensis and evaluated for fungicidal activity and levels of tumor necrosis factor (TNF-alpha) in the supernatants. F1 increased the levels of H2O2 in a similar manner to IFN-gamma. However, a synergistic effect between these two activators was not detected. On the contrary, a significant fungicidal activity was only obtained after priming with IFN-gamma plus F1. This higher activity was associated with high levels of TNF-alpha in the supernatants of the cocultures. Overall, P. brasiliensis F1 fraction induced human monocytes to release relatively high levels of TNF-alpha, which, in combination with IFN-gamma, is responsible for the activation of human monocytes for effective killing of P. brasiliensis.

Heterogeneity of apoptotic pathways and c.Jun/JNK expression in malignant gliomas.

In brain tumors, the prognostic value of apoptosis is still debated, even though recent observations are rather negative. In 50 astrocytic gliomas, apoptotic nuclei were recognized by TUNEL technique and morphology and apoptotic index (AI), mitotic index (MI) and the MI/AI ratio were calculated in proliferative areas and in areas containing perinecrotic palisadings and hypercellular centres. In proliferative areas, a positive linear correlation between MI and AI and a MI/AI ratio > I were found. The latter was < I in perinecrotic palisadings and hypercellular centres. This suggests the possibility that apoptosis is triggered respectively by cell proliferation and hypoxia. A small number of apoptotic nuclei was positive for c-Jun and JNKI, suggesting the involvement of this pathway in apoptosis as well as that of sphingomyelinase/ceramide. No relationship was found between AI and labeling indices of Bcl-2, Bcl-x, Bax, p53, APO.I/Fas. The rationale for a prognostic value of apoptosis in gliomas is thus poor.

Isolated hemangioblastoma of the filum terminale. Case report.

The filum terminale is an exceptional location for isolated hemangioblastoma. Only five cases are reported in literature, prior to the magnetic resonance imaging era. A 57-year-old man was referred to our Department with a prolonged history of progressive back pain, particularly severe when recumbent, and recurrent unilateral sciatalgia. Computed tomography demonstrated a non-homogeneous mass at L4 level. Magnetic resonance imaging displayed earliness and homogeneous enhancement of the mass with tortuous vessels above the rostral pole of the tumour, suggesting the diagnosis of a vascular tumour. The tumour was totally removed. Histological examination confirmed the hemangioblastoma diagnosis. The case indicates that hemangioblastoma, although uncommon, must be taken into consideration in cauda equina tumour diagnosis, and also emphasises the specificity of magnetic resonance features.

Bone allografts and adjuvant cisplatin for the treatment of canine appendicular osteosarcoma in 18 dogs.

The results achieved in 18 dogs following the use of frozen bone cortical allografts for limb-sparing resection of non-metastatic canine appendicular osteosarcoma are presented. Three to five cisplatin doses (70 mg/m2) were administered, starting the day after surgery. The mean and median survival times were 478 and 266 days (range 80 to 2,611 days), respectively. The survival rate was 94 per cent at three months, 78 per cent at six months, 35 per cent at 12 months, 23 per cent at 18 months and 19 per cent at 24 months; the disease-free interval was 80 to 1,246 days (mean 365 days, median 266 days). Lung metastasis developed in 55 per cent of the dogs within one year. Complications were observed in 14/18 dogs (78 per cent), comprising local recurrence (28 per cent), allograft infection (39 per cent) and implant failure (11 per cent). Despite complications, limb sparing is a useful alternative to amputation in selected cases of appendicular osteosarcoma.

The expression of Met/hepatocyte growth factor receptor gene in giant cell tumors of bone and other benign musculoskeletal tumors.

Overexpression of the hepatocyte growth factor receptor (Met/HGF receptor), a transmembrane tyrosine kinase encoded by the MET proto-oncogene, is involved in transformation and invasive behavior of human carcinomas and sarcomas. We have previously found that bone sarcomas express high levels of Met/HGF receptor while in some cases the ligand HGF is co-expressed with the receptor, activating an autocrine loop. In this study, we analyzed 40 biopsy samples of a collection of giant cell tumors and other rare benign tumors of bone for expression of the MET proto-oncogene. These included nonossifying fibromas, osteoblastomas, desmoplastic fibromas of bone, chondroblastomas, and giant cell tumors of bone. Snap frozen samples were tested for the MET and HGF gene expression by immuno-histochemistry and Western blotting with anti-MET antibodies and RT-PCR. Over 50% of all cases scored positive for MET expression being constantly positive in recurrent or locally aggressive lesions. Sporadic co-expression of the Met/HGF receptor and ligand is also demonstrated. Met/HGF receptor expression in benign bone neoplasms suggests its early involvement in sarcomagenesis.

MET oncogene aberrant expression in canine osteosarcoma.

The objective of this study was to investigate the role of the MET oncogene in canine osteosarcoma. Seven large-breed dogs affected by spontaneous skeletal osteosarcoma underwent en bloc tumor excision. Total RNA was extracted from frozen tumor samples and assessed for expression of the MET oncogene by Northern blot analysis. Five of seven biopsy samples expressed high levels of the MET oncogene; its expression in the primary tumors was comparable with that previously identified in primary osteosarcomas in humans. A lung metastasis from one of the dogs expressed MET at a higher level than did its primary tumor. Spontaneously arising osteosarcoma in dogs clinically and pathologically mimics the corresponding disease in humans. We previously demonstrated that the MET oncogene was aberrantly expressed in a high percentage of human osteosarcomas. The results of the current study also provide a molecular parallel between the tumors in dogs and humans. This in vivo model may be helpful in evaluating new strategies for therapy against osteosarcoma.

Assessment of vascularized fibular graft one year after reconstruction of the wrist after excision of a giant-cell tumour.

We report a patient in whom the distal radius was resected for a giant cell tumour and the bone defect was replaced using a vascularized proximal fibular graft. The graft was viable and hypertrophied and normal callus formed on the distal radius. Due to chronic instability of the wrist the patient underwent revision arthrodesis 1 year after resection. Microscopic studies of the epishyseal region of the fibula showed wide necrosis of the graft with active creeping substitution. Despite the good technical result of the vascularized fibular graft, the vascularization was incomplete in the proximal epiphysis. We discuss possible reasons for this.

An atypical presentation of Paget's disease in an immunocompromised individual. A case report.

Paget's disease of bone is a localized disorder of bony resorption. The mechanism underlying the development of the disease remains controversial. There is substantial evidence suggesting a genetic basis for Paget's disease in some patients. A viral etiology of Paget's disease has been advocated. A further hypothesis implicating an immunological mechanism for this disease is based on growing evidence reviewed in the text. The presented case showed clinical and X-ray features typical of a very aggressive form of Paget's disease. We hypothesize that the extreme local aggressiveness of this case was secondary to the patient's concomitant immunosuppression due to an extended therapy following renal transplant.

Distal ulnar tumours. Results of management by en bloc resection in nine patients and review of the literature.

A variety of reconstructive procedures have been suggested for stabilizing the ulnar shaft following resection of the distal ulna for tumour. We present the results of a series of nine distal ulnar tumour resections in which four different stabilization techniques were employed. We based our results on an evaluation of function, pain, motion, strength and instability. We obtained good or excellent results in seven patients treated with a soft tissue stabilization of the ulnar stump. One patient did not undergo any stabilization procedure and scored fair in our system. A further patient who required a radiocarpal arthrodesis also had an inferior result. These results suggest that soft tissue stabilization of the ulnar stump should be performed whenever possible.

Capsular replacement with synthetic mesh: effectiveness in preventing postoperative dislocation after wide resection of proximal femoral tumors and prosthetic reconstruction.

We describe a surgical technique for replacing the hip joint capsule using synthetic mesh after oncological resections of the proximal femur that resulted in gross intraoperative instability of the prosthetic reconstruction. The results of its use in 13 patients, 6 of whom also had pelvic resections, are described. These patients were selected from a total group of 88 patients undergoing proximal femoral replacement, 75 of whom did not require capsular replacement (none of these 75 patients have experienced dislocation). In the group requiring capsular reconstruction, 1 of 4 patients with bipolar hemiarthroplasty and 4 of 9 patients with total hip replacements experienced dislocation after operation. Of the dislocated total hip replacements, 1 remains chronically dislocated, and 3 were successfully stabilized by open reduction with further capsular augmentation. Given that the resections involved removal of most of the soft tissues stabilizing the hip joint, we believe that the technique of capsular reconstruction is useful in this difficult group of patients.

Acetabular osteoblastoma: description of a case.

Osteoblastoma is a slow-progressing, benign bone tumor, that is not frequently observed in clinical orthopaedics (approximately 1% of all primary bone tumors). There is predilection for the vertebrae (posterior arch), the femur, the tibia, and the cranium; it affects young subjects (from 10 to 35 years), with predilection for males (males: females = 2:1). Symptoms are not very specific, characterized essentially by moderate, discontinuous pain, that is responsive to treatment by NSAIDS; it may, at times, be asymptomatic. On radiographic assessment it is viewed as a lytic area that is rounded, greater than 2 cm in size, with unclear margins, with or without peripheral bone reaction. It is not easy to diagnose osteoblastoma, particularly if it is localized in unusual sites, such as in the pelvis. The authors present a case of osteoblastoma of the acetabular bottom in a subject aged 22 years, that was not diagnosed unrecognized for about 2 years from the onset of symptoms.

Cellular requirements for immunomodulatory effects caused by cell wall components of Paracoccidioides brasiliensis on antibody production.

In a previous study, we reported an increase in the number of immunoglobulin-secreting cells and the augmentation of antibody production (IgM and IgG3) against unrelated antigens (sheep erythrocytes or bovine serum albumin (BSA)) in mice infected with the fungus Paracoccidioides brasiliensis as well as in mice inoculated with its cell wall preparation (CW). The immunomodulatory effect of the live fungus and CW preparation was dose-dependent and mainly restricted to the i.p. inoculation simultaneously to the BSA challenge by the i.v. route. In the present study, we investigated the active component of CW preparation upon the phenotype and also the degree of activation of possible target peritoneal cells involved in those phenomena. An insoluble polysaccharide fraction (F1 fraction) mainly composed of beta-glucan and chitin, and the purified beta-glucan (BGPb) behaved as CW in the augmentation of early antibody production. The peritoneal mononuclear inflammatory cells induced by CW, F1 fraction and BGPb were highly positive to alpha-naphthyl esterase staining; released low H2O2; expressed high levels of MHC-Ia(d) molecules and produced inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and IL-6. Phenotypic analysis by flow cytometry and immunohistochemical techniques of the inflammatory cells responding to F1 fraction showed a prevalence of (CD11b/CD18, Mac-1)+ peritoneal macrophages. In addition, s.c. inoculation of F1 fraction resulted in the formation of nodular, localized and not progressive granulomatous lesions with an accumulation of (CD11b/C18)+ macrophages. Adoptive transferred Mac-1 macrophages to immunized syngeneic recipient mice were able to cause an increase in anti-BSA antibody production. These results suggest that inflammatory (CD11b/CD18)+ macrophages may be related to immunological disturbances, caused by cell wall components of P. brasiliensis.

[Primary malignant non-Hodgkin's lymphomas of the central nervous system in immunocompetent patients: diagnostic, prognostic and therapeutic criteria]. / Linfomi maligni non Hodgkin primitivi del sistema nervoso centrale nei soggetti immunocompetenti: criteri diagnostici, prognostici e terapeutici.

Primary central nervous system non-Hodgkin's malignant lymphomas (PCNSNHML) have undergone a remarkable increase in incidence in the last years. Clinically PCNSNHML may present with the symptoms that mimic many others neurological conditions. Radiologically they appear as iso or hyperdense deposits, often multiple, usually supratentorial in periventricular region. Histologically although most lesions are high grade B cell lymphomas, T cell lymphomas are being recognised with increasing frequency. Actually a substantial improvement in survival has been obtained using chemotherapy as sole treatment, eventually followed by radiotherapy for recurrences. The prognosis, however, remains poor in comparison with nodal lymphomas. It is improved by T-cell phenotype, a Karnofsky performance status more than 50%, age at onset less than 50 years and negativity for bcl-2 oncogene of neoplastic cells.