A. Zambonin Zallone

Alendronate Reduces Adhesion of Human Osteoclast-like Cells to Bone and Bone Protein-Coated Surfaces

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs. controls. The effect of ALN on the adhesion of osteoclast-like cells onto specific extracellular matrix proteins, such as bone sialoprotein-derived peptide, containing the RGD sequence, conjugated to BSA (BSP-BSA) and fibronectin (FN), was also tested. In the case of FN the treatment with ALN of protein-coated wells did not modify the percentage of cell adhesion compared with the control, whereas onto BSP-BSA the presence of ALN significantly reduced adhesion of about 40–45%, suggesting that the inhibitory effect of ALN on cell adhesion could probably be due to the interference with receptors specifically recognizing bone matrix proteins as αVβ3 integrins. Furthermore, ALN induced Ca-mediated intracellular signals in osteoclasts, triggering a 2-fold increase in intracellular calcium concentration.

Resorption of vital or devitalized bone by isolated osteoclasts in vitro

SummaryThe maintainance of resorptive capability towards vital or devitalized bone in osteoclasts isolated from the medullary bone of laying hens and cultured for five days in vitro has been investigated morphologically with the aid of light and transmission electron microscopy. Devitalized bone particles ranging in size from 50 to 100 μm, added to cultures of osteoclasts, were rapidly surrounded by the osteoclasts which, in transmission electron microscopy, showed ruffled borders and clear zones at the surfaces of contact with bone — features typical of resorptive activity. Alternatively osteoclasts were added onto the endosteal surfaces of vital or devitalized diaphyses of quail femurs after removal of the endosteal and periosteal cell layers. The results indicated that, when the vital or devitalized bone surfaces were devoid of cells, the osteoclasts adhered and resorbed bone (as confirmed by transmission electron microscopy). When vital bone of quail was cultured for 24 h before the addition of osteoclasts a new cell layer was formed; it enveloped all bone surfaces and precluded the access of osteoclasts to bone. The role of these lining cells, ultrastructurally indistinguishable from resting osteoblasts, is discussed.

Number, size and arrangement of osteoblasts in osteons at different stages of formation.

ConclusionsThe present results indicate that the appositional growth rate of the osteogenetic surfaces seems to be related to two cell parameters: a) the secreting activity of each osteoblast, which in turn appears to be a function of both cell volume and extent of the ‘secretory territory’, and b) the number of osteoblasts per unit surface of the osteogenetic front. The observed marked variability of the shape and size of osteoblasts, in relation to simply physiological variations of the appositional growth rate, suggests that the analyses of the structural responses of osteoblasts to hormones, vitamins etc. or to pathological conditions must be carried out with care.

The osteoclasts of hen medullary bone under hypocalcaemic conditions

SummaryOsteoclasts of medullary bone after several days of hypocalcaemic diet are substituted on the trabecular surface by active osteoblasts. The fate and the ultrastructure of the osteoclasts withdrawn from medullary bone surfaces in the course of a low calcium diet has been studied in serial semithin and ultrathin sections. The cytoplasmic surface of osteoclasts located in marrow compartments presents blebs and protrusions and the whole cell is often irregularly branched in several directions. A large amount of granular endoplasmic reticulum is accumulated at the cell periphery; often the cisternae are distended to form vesicles with an inner core of dense material. Osteoclasts seem to divide into mono or polynucleated smaller units.

Microdissection, Cytocochleogram and Transmission Electron Microscopy: a Technique for a Comprehensive Evaluation of Human Cochlear Pathology

A technique combining microdissection with cytocochleogram and TEM is described as a tool for studying human cochlear pathology. It is recommended in cases well studied from a clinical point of view and with a short time interval between death and fixation.

Human Osteoclast-Like Cells from Giant Cell Tumors of Bone: A New Tool for Investigating Bone Resorption and Osteoclast Biology

The effort of culturing human osteoclasts for studies on the resorptive activity have been undertaken without success in many laboratories because of the difficulty of having a sufficient number of differentiated cells from both surgery fragments or generated in vitro. To overcome this difficulty we have extensively studied the cell populations harvested from several human giant cell tumors of bone. In the literature, discordant reports concerning these tumors can be found ,some indicating a stromal nature for the transformed cells and the osteoclasts simply as reactive cells in the tissue [1, 2]. Other authors have shown that the populations obtained maintained multinuclear elements after passages [3] or that the cells, although mononucleated, were responsive to calcitonin [4]. This discrepancy can be explained if the tumors classified as osteoclastoma are two different kinds: one in which the transformed element is a stromal cell, endowed with the capability of recruiting osteoclasts and another in which the transformed cell belongs to the osteoclastic lineage. With this aim, cells from giant cell tumors of bone (GCT) have been characterized from surgical samples obtained in the course of the surgical removal of the tumor, with the informed consent of the patient. The morphological features of these cultures, together with their total and tartrate-resistant acid phosphatase activity, bone resorbing capability, and response to calciotropic hormones have been evaluated. Among all the tumors examined (16 up to now), three with a complete panel of osteoclastic features were found, whereas other three, although mononucleated , were responsive to calcitonin and capable of bone resorption. The cultures from GeT 23, 24, and 29 contained monoand multinuclear cells maintained after several passages (up to 13th until now). Multinuclear cells were originated by both fusion or endomitosis. Tartrate-resistant alkaline phosphatase (TRAP) activity histochemically evaluated was present at different degrees, but could be biochemically evaluated and always ranged around 20% of the total AP activity. Treatment with calcitonin approximately doubled the intracellular content of cAMP. Bone resorption was assayed both biochemically, utilizing prelabeled bone fragments, and morphologically, measuring the formation of resorption pits on bone slices. With both techniques, resorption was evident and in the presence of calcitonin was significantly reduced [5]. Interactions with connective tissue or bone matrixderived proteins were also studied. GCTs adhered and spread on fibronectin, osteopontin, BSPII, as in the pres-

Ultrastruetural Evaluation of the Microslicing Method for the Study of Temporal Bone Pathology

Microslices 3 mm thick from undecalcified human temporal bones were prepared with a special cutting machine and then processed for SEM and TEM in order to evaluate advantages and disadvantages of the microslicing technique for the study of the temporal bone pathology. In the examined microslices there was some mechanical distortion of the membranous labyrinth, detachment of soft tissues from bone and a considerable amount of contamination by bone dust and debris which are circulated during sectioning. For SEM the method therefore has limited value. For TEM a relatively contamination free area can be found some distance from the cutting surface of each microslice.

Hepatocyte growth factor links osteoclast-osteoblast actyvity through an autocrine-paracrine signaling

OSTEOBLAST ACTYVITY THROUGH AN AUTOCRINE PARA CRLNE SIGNALING M. Grano, F. Galimi, G. Zambonin, S. Colucei, G. Mori, E. Cottone, P.M. Comoglio, A. Zambonin 7_,allone. Institute of Human, I Orthopaedic Clinic, University of Bari Medical School, and Dept. of Biomedical Science & ontology, University of Torino. Hepatocyte Growth Factor CRGF) is a disulfide-linked haterodimer secreted by cells of mesodermal origin as an inactive single chain precorsor, that is activated to mature form by extraenllular umkinase and related protcases. The HGF receptor, the tyrosine kinase encoded by the c-MET protoeneogane (plg~r) , has been recently detected in a variety of epithelial er raesedermal derived cells. We examined if pl9lY ~ r was expressed in primary human osteoblasts and osteoclasta as well as in tumor derived osteoclast-like cell lines (GCTs) by immunofluorescence and western blotting analysis, finding a high level of expression in both cell ty~s. The treatment with HGF stimulated receptor kinase activity suggasling a potemial role of HGF in bone remodeling, thus the possible biological effects of HGF on osteoblast and osteoclast activity were evaluated. A motility response was highly evident in osteoclasts at concentration of 5 ng/ml compared with negative control, while no effect was found in osteoblasts. A dose-response stimulation of cell proliferation, evaluated by 3H-thymidinr incorporation, was found both in osteeclasts where a concentration of 7.5 ng/ml HGF induced a four-fold increase in 3Hthymidine incorporation, and in osteoblasts where a three-fold increase was already found at 2 ng/ml. The effects of HGF on ostooblast parameters as alkaline phosphatase activity and osteocalcin production were evaluated, demoostrating an inhibitory effect already visible at low dosages. lutraecllular signals as variation in intracellniar calcium concentration 2+ 2+ [Ca ]i were also iovestigated, showing an HGF induced [Ca li increase in osteeclasts and in GCT cells and ne effects io esteoblasls, c-Src kinase activity was evaluated following stimulation by the HGF iu osteoclast-like GCT cells. Results showed a 2-fold increase in pp60-c-Sre kinase in the cells treated with HGF. Moreover results obtained with western blot technique indicate that osteoclasts can produce HGF suggesting an autucrine-garacrine activity and the possible identification of HGF as a coopling factor of osteoclast and osteoblast activily. These data, together with the already demonstrated angiogenic activity of HGF, lel us hipothesizr a prominent role of this growth factor during skelelogenesis and bone remodeling

Degeneration patterns in the organ of Corti and spiral lamina.

The relation between organ of Corti degeneration and radial nerve fibre degeneration in the osseous spiral lamina was studied in two human cochleas.