Bone Quality Changes In 3 Cases Of Hypophosphatasia: A Ftiri Study

International audienc

Stable Incretin Mimetics Counter Rapid Deterioration of Bone Quality in Type 1 Diabetes Mellitus.

AIMS: Type 1 diabetes mellitus is associated with a high risk for bone fractures. Although bone mass is reduced, bone quality is also dramatically altered in this disorder. However, recent evidences suggest a beneficial effect of the glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) pathways on bone quality. The aims of the present study were to conduct a comprehensive investigation of bone strength at the organ and tissue level; and to ascertain whether enzyme resistant GIP or GLP-1 mimetic could be beneficial in preventing bone fragility in type 1 diabetes mellitus. MATERIALS AND METHODS: Streptozotocin-treated mice were used as a model of type 1 diabetes mellitus. Control and streptozotocin-diabetic animals were treated for 21 days with an enzymatic-resistant GIP peptide ([D-Ala2]GIP) or with liraglutide (each at 25 nmol/kg bw, ip). Bone quality was assessed at the organ and tissue level by microCT, qXRI, 3-point bending, qBEI, nanoindentation and Fourier-transform infrared microspectroscopy. RESULTS: [D-Ala2]GIP and liraglutide treatment did prevent loss of whole bone strength and cortical microstructure in the STZ-injected mice. However, tissue material properties were significantly improved in STZ-injected animals following treatment with [D-Ala(2) ]GIP or liraglutide. CONCLUSIONS: Treatment of STZ-diabetic mice with [D-Ala2]GIP or liraglutide was capable of significantly preventing deterioration of the quality of the bone matrix. Further studies are required to further elucidate the molecular mechanisms involved and to validate whether these findings can be translated to human patients. This article is protected by copyright. All rights reserved

Technical aspects: how do we best prepare bone samples for proper histological analysis?

Histological analysis of bone is a critical step for the diagnosis of malignancies. It allows direct identification of malignant cells inside marrow spaces in case of bone metastases or hematological disorders. Bone biopsy is superior to marrow aspiration because the microarchitecture of the bone marrow is preserved, a parameter that is especially important in hematological disorders. Because marrow cells are in direct contact with bone cells (lining cells, osteoblasts, osteoclasts, and their precursors), an abnormal bone remodeling rate has been described in a variety of malignant cell proliferations when developing and expanding inside marrow spaces. Bone cells elaborate and synthesize a variety of cytokines acting on hematological precursors (e.g., M-CSF)1 and malignant cells release other cytokines active on bone remodeling2–4: it is likely that bone changes are almost always associated with bone marrow alterations and vice versa. Histomorphometric analysis is a powerful tool in the evaluation of bone remodeling in metabolic bone diseases and was also successfully applied to hematological disorders and metastases from solid tumors5,6. Bone histomorphometry is a powerful method in the early diagnosis of B-cell malignancies, and smoldering myeloma or lymphomas can be characterized in patients with a monoclonal gammopathy of undetermined significance (MGUS) several years before the tumor has shown clinical expression. Bone histomorphometry is also useful in animal models of cancer bone lesions, since it permits a precise evaluation of the bone remodeling changes induced by tumor cells7–9. However, bone histomorphometry must be done on undecalcified bone sections which allow a perfect identification of osteoid tissue (the unmineralized bone matrix recently synthesized by osteoblasts), a precise identification of osteoclasts (by using histoenzymatic detection) and histodynamic analyses (after a double tetracycline labeling in humans or using a variety of other fluorochromes in the animal). These methods cannot be used on decalcified and paraffin embedded bone, since decalcification abolishes the osteoid/bone matrix differential staining and removes the fluorochrome labels, and hot paraffin embedding destroys enzyme activities. However, decalcification and paraffin remain useful for immunohistochemistry, which is difficult and hazardous on plastic sections. The main disadvantage of polymer embedding was formerly the prolonged time for preparing bone specimens (several months when polyester resins were used). With the development of histological techniques, it is now possible to have polymer embedding methods that are as fast as conventional paraffin methods. The following techniques have been developed and improved in our laboratory during the last two decades and used on more than 3000 human bone biopsies and a large number of animal studies performed in a variety of animal species (for example, mouse, rat, chicken, dog, goat, sheep, pig)

Osteopontin is an argentophilic protein in the bone matrix and in cells of kidney convoluted tubules.

International audienceNucleolar organising regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23). They are identified by silver staining at low pH. The method also reveals osteocyte canaliculi and cement lines and granules in the cytoplasm of kidney cells in locations that mimic osteopontin distribution. Human bone and kidney sections, benign and lymphomatous pleural effusions were processed for silver staining to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. In pleural effusions, AgNORs were found increased in the nuclei of lymphoma cells. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. NOR proteins and osteopontin are proteins containing aspartic acid rich repeats that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections. AgNOR is a useful histochemical method to identify osteopontin in bone sections

Osteopontin is histochemically detected by the AgNOR acid-silver staining

Silver nitrate staining of decalcified bone sections is known to reveal osteocyte canaliculi and cement lines. Nucleolar Organising Regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23) that can be identified by silver staining at low pH. The aim of this study was to clarify the mechanism explaining why AgNOR staining also reveals osteocyte canaliculi. Human bone and kidney sections were processed for silver staining at light and electron microscopy with a modified method used to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. Protein extraction was done in the renal cortex and decalcified bone and the proteins were separated by western blotting. Purified hOPN was also used as a control. Proteins were electro-transferred on polyvinylidene difluoride membranes and stained for AgNOR proteins. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. Ag staining of membranes at low pH revealed bands for NOR proteins and 56 KDa (kidney), 60KDa (purified hOPN) and 75 KDa (bone) bands that corresponded to osteopontin. NOR proteins and osteopontin are proteins containing aspartic acid rich regions that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections or membranes