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Marzia FERRETTI

Professore Associato
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze,sede Istituti Anatomici (area Policlinico)


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Pubblicazioni

2022 - Expression and localization of Phosphoinositide-specific Phospholipases C in cultured, differentiating and stimulated human osteoblasts [Articolo su rivista]
Casoni Sara, Daisy; Romanelli, Alessia; Checchi, Marta; Truocchio, Serena; Ferretti, Marzia; Palumbo, Carla; LO VASCO, VINCENZA RITA
abstract

The osteoblasts contribute to bone homeostasis maintaining the bone mass, and intervene in bone injuries repair. The limited number of available therapeutic agents promoting osteogenesis aroused the greatest interest in the control of osteoblasts’ activity. Insights in the events leading to the proliferation and differentiation of osteoblasts might allow uncover potential molecular targets to control the complex mechanisms underlying bone remodeling. Oscillations of calcium act crucially during this remodeling, affecting both the differentiation and proliferation of osteoblasts. Signal transduction pathways contribute to the differentiation and metabolic activities of osteoblasts, with special regard to calcium-related signaling, including the Phosphoinositide (PI) pathway and related Phospholipases C (PLCs). In order to evaluate the role of PLC enzymes’ family in human osteoblasts (HOBs), we analyzed the expression of PLC genes and the localization of PLC enzymes in cultured HOBs and in in vitro differentiating HOBs after 3, 10, 17 and 23 days, and in HOBs stimulated with Lipopolysaccharide, which affects the differentiation of osteoblasts, after 3, 6, 24 and 48 hours. Our results confirm the transcription of most PLC genes and the presence of a number of PLC enzymes in HOBs, differently localized in the nucleus, in the cytoplasm or both, as well as in cell protrusions. The localization of PLC enzymes within the cell suggests the activation of both the PI nuclear and of the cytoplasmic cycle in HOBs. Depending on the experimental conditions, transcripts of splicing variants of selected PLC genes were detected and the localization of most PLC enzymes varied, with special regard to enzymes belonging to the PLC ,  and  sub-families. Further studies addressed to elucidate the complex network involving the signal transduction of PLCs might provide further insights into the complex signal transduction network in bone remodeling, also offering the opportunity to identify promising molecular targets.


2022 - From morphological basic research to proposals for regenerative medicine through a translational perspective [Articolo su rivista]
Checchi, Marta; Stanzani, Virginia; Truocchio, Serena; Corradini, Matteo; Ferretti, Marzia; Palumbo, Carla
abstract


2021 - Static Osteogenesis versus Dynamic Osteogenesis: A Comparison between Two Different Types of Bone Formation [Articolo su rivista]
Ferretti, Marzia; Palumbo, Carla
abstract

In contrary to what has traditionally been believed, bone formation can occur through two different types of osteogenesis: static (SO) and dynamic (DO) osteogenesis, which are thus named because the former is characterized by pluristratified cords of unexpectedly stationary osteoblasts which differentiate at a fairly constant distance from the blood capillaries and transform into osteocytes without moving from the onset site, while the latter is distinguished by the well-known typical monostratified laminae of movable osteoblasts. The two types of osteogenesis differ in multiple aspects from both structural and functional viewpoints. Besides osteoblast arrangement, polarization, and motion, SO and DO differ in terms of time of occurrence (first SO and later DO), conditioning factors to which they are sensitive (endothelial-derived cytokines or mechanical loading, respectively), distribution of osteocytes to which they give rise (haphazard or ordered in planes, respectively), the collagen texture resulting from the different deposition types (woven or lamellar, respectively), the mechanical properties of the bone they form (poor for SO due to the high cellularity and woven texture and good for DO since osteocytes are located in more suitable conditions to perceive loading), and finally the functions of each, i.e., SO provides a preliminary rigid scaffold on which DO can take place, while DO produces bone tissue according to mechanical/metabolic needs.


2021 - The anatomy and its variants [Capitolo/Saggio]
Palumbo, Carla; Ferretti, Marzia; LO VASCO, VINCENZA RITA
abstract

Visceral and renal artery aneurysms are rare but life-threatening pathologies. Many techniques have been proposed for their treatment, and both open surgery and endovascular strategies have proven to be safe and effective. Many specialists can be involved in the treatment of these aneurysms, from vascular surgeons to interventional radiologists, and the knowledge of every different possible approach is fundamental to offer the patient the best outcome. In this book, all the aspects of visceral and renal artery aneurysms have been widely discussed, from epidemiology and anatomical variations to surgical approaches and materials. To conclude, the authors reported a large series of case reports from different experts in order to obtain a better vision of the real-world practice on this topic.


2021 - The Osteocyte: From “Prisoner” to “Orchestrator” [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia
abstract

Osteocytes are the most abundant bone cells, entrapped inside the mineralized bone matrix. They derive from osteoblasts through a complex series of morpho-functional modifications; such modifications not only concern the cell shape (from prismatic to dendritic) and location (along the vascular bone surfaces or enclosed inside the lacuno-canalicular cavities, respectively) but also their role in bone processes (secretion/mineralization of preosseous matrix and/or regulation of bone remodeling). Osteocytes are connected with each other by means of different types of junctions, among which the gap junctions enable osteocytes inside the matrix to act in a neuronal-like manner, as a functional syncytium together with the cells placed on the vascular bone surfaces (osteoblasts or bone lining cells), the stromal cells and the endothelial cells, i.e., the bone basic cellular system (BBCS).Within the BBCS, osteocytes can communicate in two ways: by means of volume transmission and wiring transmission, depending on the type of signals (metabolic or mechanical, respectively) received and/or to be forwarded. The capability of osteocytes in maintaining skeletal and mineral homeostasis is due to the fact that it acts as a mechano-sensor, able to transduce mechanical strains into biological signals and to trigger/modulate the bone remodeling, also because of the relevant role of sclerostin secreted by osteocytes, thus regulating different bone cell signaling pathways. The authors want to emphasize that the present review is centered on the morphological aspects of the osteocytes that clearly explain their functional implications and their role as bone orchestrators.


2020 - Bone Healing Evaluation Following Different Osteotomic Techniques in Animal Models: A Suitable Method for Clinical Insights [Articolo su rivista]
Anesi, Alexandre; Di Bartolomeo, Mattia; Pellacani, Arrigo; Ferretti, Marzia; Cavani, Francesco; Salvatori, Roberta; Nocini, Riccardo; Palumbo, Carla; Chiarini, Luigi
abstract

Osteotomy is a common step in oncological, reconstructive, and trauma surgery. Drilling and elevated temperature during osteotomy produce thermal osteonecrosis. Heat and associated mechanical damage during osteotomy can impair bone healing, with consequent failure of fracture fixation or dental implants. Several ex vivo studies on animal bone were recently focused on heating production during osteotomy with conventional drill and piezoelectric devices, particularly in endosseous dental implant sites. The current literature on bone drilling and osteotomic surface analysis is here reviewed and the dynamics of bone healing after osteotomy with traditional and piezoelectric devices are discussed. Moreover, the methodologies involved in the experimental osteotomy and clinical studies are compared, focusing on ex vivo and in vivo findings.


2020 - WISP-2 expression induced by Teriparatide treatment affects in vitro osteoblast differentiation and improves in vivo osteogenesis [Articolo su rivista]
Smargiassi, A.; Bertacchini, J.; Checchi, M.; Poti, F.; Tenedini, E.; Montosi, G.; Magaro, M. S.; Amore, E.; Cavani, F.; Ferretti, M.; Grisendi, G.; Maurel, D. B.; Palumbo, C.
abstract

The Osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances occurring during bone remodeling, caused by hormone perturbations or by mechanical loading alterations, can induce bone pathologies such as osteoporosis. Recently, the active fraction of parathormone, PTH (1-34) or Teriparatide (TPTD), was chosen as election treatment for osteoporosis. The effect of such therapy is dependent on the temporal manner of administration. The molecular reasons why the type of administration regimen is so critical for the fate of bone remodeling are numerous and not yet well known. Our study attempts to analyze diverse signaling pathways directly activated in osteocytes upon TPTD treatment. By means of gene array analysis, we found many molecules upregulated or downregulated in osteocytes. Later, we paid attention to Wisp-2, a protein involved in the Wnt pathway, that is secreted by MLO-Y4 cells and increases upon TPTD treatment and that is able to positively influence the early phases of osteogenic differentiation. We also confirmed the pro osteogenic property of Wisp-2 during mesenchymal stem cell differentiation into the preliminary osteoblast phenotype. The same results were confirmed with an in vivo approach confirming a remarkable Wisp-2 expression in metaphyseal trabecular bone. These results highlighted the anabolic roles unrolled by osteocytes in controlling the action of neighboring cells, suggesting that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation.


2019 - Bi-layered collagen nano-structured membrane prototype collagen matrix CM-10826 for oral soft tissue regeneration: an in vivo ultrastructural study on 13 patients [Articolo su rivista]
De Santis, D.; Gelpi, F.; Castellani, R.; Palumbo, C.; Ferretti, M.; Zanotti, G.; Zotti, F.; Montagna, L.; Luciano, U.; Marconcini, S.; Tacchino, U.; Manuelli, M.; Nocini, R.; Nocini, P. F.; Albanese, Maria Cristina Nicoletta
abstract

A new developed collagen matrix CM-10826 (CM) of porcine origin designed to be used as oral soft tissue substitute was investigated before and after implantation by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). In a case series biopsy specimens were harvested from thirteen patients at 10, 20, 30, 43 days after abutment surgery for uncovering dental implants. The in vivo histological evaluations of each patient were performed via micro-coring of newly formed oral mucosa in the area covered by CM (test side) or left uncovered (control). Results showed that CM can be integrated in connective and epithelial tissues within 10 days, can be completely resorbed within 20 days and it is able to reduce inflammatory infiltrates and to stimulate both fibroblast/epithelial cell proliferation and neo-angiogenesis. Generally it seems to be superior in promoting soft tissue healing compared to that induced by secondary intention healing. Furthermore, it is able to act as a scaffold for soft-tissue regeneration, allowing the proliferation of keratinocytes from the wound edges and favoring neovascularization and growth of connective tissue in the mesh of porous layer. It appears that a CM might function in oral surgery as a substitute for autologous grafts and to avoid secondary intention healing in soft tissue defects.


2019 - Interaction among Calcium Diet Content, PTH (1-34) Treatment and Balance of Bone Homeostasis in Rat Model: The Trabecular Bone as Keystone [Articolo su rivista]
Ferretti, Marzia; Cavani, Francesco; Roli, Laura; Checchi, Marta; Magarò, Maria Sara; Bertacchini, Jessika; Palumbo, Carla
abstract

The present study is the second step (concerning normal diet restoration) of the our previous study (concerning the calcium-free diet) to determine whether normal diet restoration, with/without concomitant PTH (1-34) administration, can influence amounts and deposition sites of the total bone mass. Histomorphometric evaluations and immunohistochemical analysis for Sclerostin expression were conducted on the vertebral bodies and femurs in the rat model. The final goals are (i) to define timing and manners of bone mass changes when calcium is restored to the diet, (ii) to analyze the different involvement of the two bony architectures having different metabolism (i.e., trabecular versus cortical bone), and (iii) to verify the eventual role of PTH (1-34) administration. Results evidenced the greater involvement of the trabecular bone with respect to the cortical bone, in response to different levels of calcium content in the diet, and the effect of PTH, mostly in the recovery of trabecular bony architecture. The main findings emerged from the present study are (i) the importance of the interplay between mineral homeostasis and skeletal homeostasis in modulating and guiding bone's response to dietary/metabolic alterations and (ii) the evidence that the more involved bony architecture is the trabecular bone, the most susceptible to the dynamical balance of the two homeostases.


2019 - Trabecular Bone as Keystone for the Interplay Among Calcium Diet Content, PTH(1-34) Treatment and Balance of Bone Homeostases in Rat Model [Capitolo/Saggio]
Ferretti, Marzia; Cavani, Francesco; Checchi, Marta; Magaro', MARIA SARA; Amore, Emanuela; Bertacchini, Jessika; Palumbo, Carla
abstract

The present study aims to determine whether normal-diet restoration, with/without concomitant PTH(1-34) administration, can influence amounts and deposition sites of the total bone mass. Histomorphometric evaluations and immunohistochemical analysis for Sclerostin expression were conducted on the vertebral bodies and femurs in the rat model. Final goals 1) to define timing and manners of bone mass changes when calcium is restored to the diet; 2) to analyze the different involvement of the two bony architectures having different metabolism (i.e. trabecular versus cortical bone); 3) to verify the eventual role of PTH(1-34) administration. Results evidenced the greater involvement of the trabecular bone with respect to the cortical bone, in response to differing levels of calcium content in the diet, and the effect of PTH, mostly in the recovery of trabecular bony architecture. The main findings emerged are: i) the importance of the interplay between mineral homeostasis and skeletal homeostasis in modulating and guiding bone’s response to dietary/metabolic alterations and ii) the evidence that the more involved bony architecture is trabecular bones, the most susceptible to the dynamical balance of mineral and skeletal homeostasis.


2018 - Angiogenic and inflammatory potential of Scleral Ossicles, novel natural biomaterials for bone regeneration [Abstract in Rivista]
Checchi, Marta; Bertacchini, Jessika; Magaro', MARIA SARA; Ferretti, Marzia; Sola, Antonella; Bisi, Francesca; Messori, Massimo; Ribatti, Domenico; Maurel, Delphine; Palumbo, Carla
abstract

The aim of this work is the analysis of the angiogenic and inflammatory potential of the Scleral Ossicles (SOs), already analysed by the structural viewpoint, and the development of a functionalized-SOs-construct. The final goal is to improve the healing of critical-size bone fractures.


2018 - Interaction between mineral and skeletal homeostasis in rats fed different calcium content diets with/without PTH (1-34) [Abstract in Rivista]
Ferretti, Marzia; Cavani, Francesco; Bertacchini, Jessika; Checchi, Marta; Magaro', MARIA SARA; Palumbo, Carla
abstract

Aim of the study is to analyze how mineral and skeletal homeostases influence both the bone loss due to calcium-diet deprivation and the successive bone mass recovery after calcium-diet restoration, with/without concomitant PTH(1-34) administration.


2018 - Role of osteocytes in myeloma bone disease: Anti-sclerostin antibody as new therapeutic strategy [Articolo su rivista]
Toscani, Denise; Bolzoni, Marina; Ferretti, Marzia; Palumbo, Carla; Giuliani, Nicola
abstract

Osteocytes are terminally differentiated cells of the osteoblast lineage. They are involved in the regulation of bone remodeling by increasing osteoclast formation or decreasing bone formation by the secretion of the osteoblast inhibitor sclerostin. Monoclonal antibody anti-sclerostin, Romosozumab, has been developed and tested in clinical trials in patients with osteoporosis. In the last years, the role of osteocytes in the development of osteolytic bone lesions that occurs in multiple myeloma, have been underlined. Myeloma cells increase osteocyte death through the up-regulation of both apoptosis and autophagy that, in turn, triggers osteoclast formation, and activity. When compared to healthy controls, myeloma patients with bone disease have higher osteocyte cell death, but the treatment with proteasome inhibitor bortezomib has been shown to maintain osteocyte viability. In preclinical mouse models of multiple myeloma, treatment with blocking anti-sclerostin antibody increased osteoblast numbers and bone formation rate reducing osteolytic bone lesions. Moreover, the combination of anti-sclerostin antibody and the osteoclast inhibitor zoledronic acid increased bone mass and fracture resistance synergistically. However, anti-sclerostin antibody did not affect tumor burden in vivo or the efficacy of antimyeloma drugs in vitro. Nevertheless, the combination therapy of antisclerostin antibody and the proteasome inhibitor carfilzomib, displayed potent anti-myeloma activity as well as positive effects on bone disease in vivo. In conclusion, all these data suggest that osteocytes are involved in myeloma bone disease and may be considered a novel target for the use of antibody-mediated anti-sclerostin therapy also in multiple myeloma patients.


2018 - Structural and ultrastructural analyses of bone regeneration in rabbit cranial osteotomy: Piezosurgery versus traditional osteotomes. [Articolo su rivista]
Anesi, Alexandre; Ferretti, Marzia; Cavani, Francesco; Salvatori, Roberta; Bianchi, Michele; Alessandro, Russo; Chiarini, Luigi; Palumbo, Carla; Bianchi, Michele
abstract

Clinical advantages of piezosurgery have been already proved. However, few investigations have focused on the dynamics of bone healing. The aim of this study was to evaluate, in adult rabbits, bone regeneration after cranial linear osteotomies with two piezoelectrical devices (Piezosurgery® Medical - PM and Piezosurgery® Plus - PP), comparing them with conventional rotary osteotomes (RO). PP was characterized by an output power three times higher than PM. Fifteen days after surgery, histomorphometric analyses showed that the osteotomy gap produced with PM and PP was about half the size of that produced by RO, and in a more advanced stage of recovery. Values of regenerated bone area with respect to the total osteotomy area were about double in PM and PP samples compared with RO ones, while the number of TRAP-positive (tartrate-resistant acid phosphatase positive) osteoclasts per linear surface showed a significant increase, suggesting greater bone remodelling. Under scanning electron microscopy, regenerated bone displayed higher cell density and less mineralized matrix compared with pre-existent bone for all devices used. Nanoindentation tests showed no changes in elastic modulus. In conclusion, PM/PP osteotomies can be considered equivalent to each other, and result in more rapid healing compared with those using RO.


2017 - Bi-layered collagen nano-structured membrane prototype collagen matrix 10826® for soft tissue regeneration in rabbits: an in vivo ultra-structural study of the early healing phase. [Articolo su rivista]
De Santis, D; Menchini Fabris, Gb; Lotti, J; Palumbo, C; Ferretti, M; Castellani, R; Lotti, T; Zanotti, G; Gelpi, F; Covani, C; Nocini, Pf
abstract

Collagen Matrix (CM) 10826 is a nanostructured bi-layered collagen membrane obtained from type I and III porcine collagen, which in vitro has shown to have the potential to be a substitute and/or stimulant for soft oral tissue regeneration. The objective of this study was to evaluate the in vivo potential and safety of this membrane for soft tissue regeneration in the early stage of wound healing. Two soft tissue wounds (test and control) were created on the back skin of 5 rabbits (female New Zealand White Rabbits specific pathogen free). All wounds were protected by a special poly-tetra-fluoro-ethylene (PTFE) healing camera. On each rabbit on the test side CM-10826 was used, while on the control side conventional treatment (an autologous pedicle graft) was performed. The healing process was observed clinically after 2 and 6 days, and Magnetic Resonance Imaging (MRI) was performed after this period. After 7 days, animals were sacrificed and specimens were analyzed with light optic microscopy (LM), Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). These in vivo trials on rabbits confirmed that CM-10826 is well tolerated, without signs of histological inflammatory reaction and proved to be able to accelerate the spontaneous repair of the skin defect taken as the control. The light-optic and ultra-microscopy of serial biopsies showed that the new matrix is biocompatible and is able to function as a scaffold inducing soft tissue regeneration. In conclusion this study demonstrates that CM-10826 promote early soft tissue regeneration and suggests it is a potential constituent for human autologous keratinocytes seeded derma bioequivalent. It protects the wound from injuries and bacterial contamination accelerating healing process. As a clinical relevance, we consider that the quality of life of patients will be improved avoiding the use of major autologous grafts, reducing the hospitalization time and morbidity.


2017 - Biocompatibility Analyses of Al₂O₃-Treated Titanium Plates Tested with Osteocyte and Fibroblast Cell Lines [Articolo su rivista]
Smargiassi, Alberto; Bertacchini, Jessika; Checchi, Marta; Cavani, Francesco; Ferretti, Marzia; Palumbo, Carla
abstract

Osseointegration of a titanium implant is still an issue in dental/orthopedic implants durable over time. The good integration of these implants is mainly due to their surface and topography. We obtained an innovative titanium surface by shooting different-in-size particles of Al₂O₃ against the titanium scaffolds which seems to be ideal for bone integration. To corroborate that, we used two different cell lines: MLO-Y4 (murine osteocytes) and 293 (human fibroblasts) and tested the titanium scaffolds untreated and treated (i.e., Al₂O₃ shot-peened titanium surfaces). Distribution, density, and expression of adhesion molecules (fibronectin and vitronectin) were evaluated under scanning electron microscope (SEM) and confocal microscope (CM). DAPI and fluorochrome-conjugated antibodies were used to highlight nuclei, fibronectin, and vitronectin, under CM; cell distribution was analyzed after gold-palladium sputtering of samples by SEM. The engineered biomaterial surfaces showed under SEM irregular morphology displaying variously-shaped spicules. Both SEM and CM observations showed better outcome in terms of cell adhesion and distribution in treated titanium surfaces with respect to the untreated ones. The results obtained clearly showed that this kind of surface-treated titanium, used to manufacture devices for dental implantology: (i) is very suitable for cell colonization, essential prerequisite for the best osseointegration, and (ii) represents an excellent solution for the development of further engineered implants with the target to obtain recovery of stable dental function over time.


2017 - Expression and functional proteomic analyses of osteocytes from Xenopus laevis tested under mechanical stress conditions: preliminary observations on an appropriate new animal model [Articolo su rivista]
Bertacchini, Jessika; Benincasa, Marta; Checchi, Marta; Cavani, Francesco; Smargiassi, Alberto; Ferretti, Marzia; Palumbo, Carla
abstract

Hitherto, the role of the osteocyte as transducer of mechanical stimuli into biological signals is far from settled. In this study, we used an appropriate model represented by the cortex of Xenopus laevis long bone diaphysis lacking (unlike the mammalian one) of vascular structures and containing only osteocytes inside the bone matrix. These structural features allow any change of protein profile that might be observed upon different experimental conditions, such as bone adaptation to stress/mechanical loading, to be ascribed specifically to osteocytes. The study was conducted by combining ultrastructural observations and two-dimensional electrophoresis for proteomic analysis. The osteocyte population was extracted from long bones of lower limbs of amphibian skeletons after different protocols (free and forced swimming). The experiments were performed on 210 frogs subdivided into five trials, each including free swimming frogs (controls) and frogs submitted to forced swimming (stressed). The stressed groups were obliged to swim (on movable spheres covering the bottom of a pool on a vibrating plate) continuously for 8 h, and killed 24 h later along with the control groups. Long bones free of soft tissues (periosteum, endosteum and bone marrow), as well as muscles of posterior limbs, were processed and analyzed for proteins differentially expressed or phosphorylated between the two sample groups. The comparative analysis showed that protein phosphorylation profiles differ between control and stressed groups. In particular, we found in long bones of stressed samples that both Erk1/2 and Akt are hyperphosphorylated; moreover, the different phosphorylation of putative Akt substrates (recognized by specific Akt phosphosubstrates-antibody) in stressed vs. control samples clearly demonstrated that Akt signaling is boosted by forced swimming (leading to an increase of mechanical stress) of amphibian long bones. In parallel, we found in posterior limb muscles that the expression of heat shock protein HSP27 and HSP70 stress markers increased upon the forced swimming condition. Because the cortexes of frog long bones are characterized by the presence of only osteocytes, all our results establish the suitability of the X. laevis animal model to study the bone response to stress conditions mediated by this cell type and pave the way for further analysis of the signaling pathways involved in these signal transduction mechanisms.


2017 - Morphological Study: Ultrastructural Aspects of Articular Cartilage and Subchondral Bone in Patients Affected by Post-Traumatic Shoulder Instability [Articolo su rivista]
Baudi, Paolo; Catani, Fabio; Rebuzzi, Manuela; Ferretti, Marzia; Smargiassi, Alberto; Campochiaro, Gabriele; Serafini, Fabio; Palumbo, Carla
abstract

Post-traumatic shoulder instability is a frequent condition in active population, representing one of most disabling pathologies, due to altered balance involving joints. No data are so far available on early ultrastructural osteo-chondral damages, associated with the onset of invalidating pathologies, like osteoarthritis-OA. Biopsies of glenoid articular cartilage and sub-chondral bone were taken from 10 adult patients underwent arthroscopic stabilization. Observations were performed under Transmission Electron Microscopy-TEM in tangential, arcuate and radial layers of the articular cartilage and in the sub-chondral bone. In tangential and arcuate layers chondrocytes display normal and very well preserved ultrastructure, probably due to the synovial liquid supply; otherwise, throughout the radial layer (un-calcified and calcified) chondrocytes show various degrees of degeneration; occasionally, in the radial layer evidences of apoptosis/autophagy were also observed. Concerning sub-chondral bone, osteocytes next to the calcified cartilage also show signs of degeneration, while osteocytes farther from the osteo-chondral border display normal ultrastructure, probably due to the bone vascular supply. The ultrastructural features of the osteo-chondral complex are not age-dependent. This study represents the first complete ultrastructural investigation of the articular osteo-chondral complex in shoulder instability, evaluating the state of preservation/viability of both chondrocytes and osteocytes throughout the successive layers of articular cartilage and sub-chondral bone. Preliminary observations here collected represent the morphological basis for further deepening of pathogenesis related to shoulder instability, enhancing the relationship between cell shape and microenvironment; in particular, they could be useful in understanding if the early surgical treatment in shoulder instability could avoid the onset of OA.


2017 - Osteocytes signaling events induced by intermittent vs continuous Teriparatide treatment affect in vitro osteoblast differentiation and mineralization [Abstract in Rivista]
Bertacchini, Jessika; Smargiassi, Alberto; Checchi, Marta; Tenedini, Elena; Montosi, Giuliana; Vinet, Jonathan; Ferretti, Marzia; Palumbo, Carla
abstract

PTH(1-34), also known as Teriparatide, is an active anabolic drug used in the treatment of some forms of osteoporosis and occasionally exploited to speed fracture healing. The effect of such therapies are dependent on the type of administration, in fact it has been largely demonstrated that a short administration of Teriparatide (also called intermittent) increases the bone mass, meanwhile a long administration of the same agent (known as continuous) leads to an increased resorption. The molecular reason why the type of administration is so critical for the fate of the bone remodeling is still largely unknown but it is probably due to the fact that it affects several signaling pathways and alters the biological activity of a cohort of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. In the present work, we firstly focused the attention on molecular events induced by intermittent vs continuous Teriparatide treatment in a well-known osteocytes in vitro model, the MLO-Y4 cells. By the use of a gene array platform, we found many molecules upregulated or downregulated depending on the the temporal administration modes, suggesting that the drug affects in diverse manner the osteocytes related signaling pathways. In particular, we paid attention to Wisp-2, a protein of the Wnt pathway that has been demonstrated to be able to interact and influence the differentiation of osteoblasts into osteocytes and their mineralization. Secondly, through the mineralization assay, we analyzed the functional effects, involving the differentiation of osteoblast IDG-SW3 cell line, upon the conditioning culture with MLO-Y4 medium, that were pre-treated with short and long time administration of Teriparatide. These findings, consistent with the crucial role performed by osteocytes on osteoblast differentiation, clarify the molecular events downstream the short treatment with Teriparatide, suggesting that the perturbation of certain signaling patwhays, such as the Wnt pathway, is crucial for the positive regulation of bone formation.


2017 - PTH(1-34) effects on repairing experimentally drilled holes in rat femur: novel aspects – qualitative vs. quantitative improvement of osteogenesis [Articolo su rivista]
Cavani, Francesco; Ferretti, Marzia; Smargiassi, Alberto; Palumbo, Carla
abstract

The timetable of effects on bone repair of the active fraction-parathyroid hormone, PTH(1-34), was analytically investigated from the morphometric viewpoint in 3-month-old male Sprague-Dawley rats, whose femurs were drilled at mid-diaphyseal level (transcortical holes). The animals were divided into groups with/without PTH(1-34) administration, and sacrificed at different times (10, 28, 45 days after surgery). The observations reported here need to be framed in the context of our previous investigations regarding bone histogenesis (Ferretti et al. Anat Embryol. 2002; 206: 21–29) in which we demonstrated the occurrence of two successive bone-forming processes during both skeletal organogenesis and bone repair, i.e. static and dynamic osteogenesis: the former (due to stationary osteoblasts, haphazardly grouped in cords) producing preliminary bad quality trabecular bone, the latter (due to typical polarized osteoblasts organized in ordered movable laminae) producing mechanically valid bone tissue. The primary function of static osteogenesis is to provide a rigid scaffold containing osteocytes (i.e. mechano-sensors) for osteoblast laminae acting in dynamic osteogenesis. In the present work, histomorphometric analysis revealed that, already 10 days after drilling, despite the holes being temporarily filled by the same amount of newly formed trabecular bone by static osteogenesis independently of the treatment, the extent of the surface of movable osteoblast-laminae (covering the trabecular surface) was statistically higher in animals submitted to PTH(1-34) administration than in control ones; this datum strongly suggests the effect of PTH(1-34) alone in anticipating the occurrence of dynamic osteogenesis involved in the production of good quality bone (with more ordered collagen texture) more suitable for loading. This study could be crucial in further translational clinical research in humans for defining the best therapeutic strategies to be applied in recovering severe skeletal lesions, particularly as regards the time of PTH(1-34) administration.


2017 - Scleral ossicles as natural biomaterials on which vascular-like network is promoted from Mouse Aortic Endothelial cells (MAECs): preliminary results [Abstract in Rivista]
Checchi, Marta; Grisendi, Giulia; Bertacchini, Jessika; Magaro', MARIA SARA; Ferretti, Marzia; Benincasa, Marta; Sena, Paola; Cavani, Francesco; Palumbo, Carla
abstract

When a severe fracture is difficult to self-recovered, it is defined as “critical-size” bone defect. Till now, many efforts have been made by the tissue engineering (TE) to generate scaffolds suitable for recovering of this type of fracture, but the main obstacle remains the lack of an appropriate vascularization of the scaffolds. In the field of the regenerative medicine, the TE has developed many different biomaterials, with various features and peculiar functions, to be used in combination with cells and growth factors, in the generation of specialized constructs. Our proposal of natural scaffolds useful to obtain complex constructs concerns peculiar bony chips extracted from the eye bulb of adult chickens: the scleral ossicles (SOs). This proposed model is interesting because once SOs reach the definitive size in the adult animal, they are devoted only to mechanical stereotyped stress for their lifetime so that the activation of the bone remodelling should be avoided and, to do this, the osteocytes undergo massive apoptosis, making the ossicles like decellularized bones [1]. The novelty of our proposal is that the scaffolds do not require surface treatment (like further matrix deposition on the SO surface) since they are characterized, like all bones, by the well-known organic components such as type I-collagen fibres, proteoglycans and glycoproteins. The latter, for example, play the role of adhesion proteins and therefore can mediate the adhesion of the endothelial cells that should develop the vascular network. Our final goal is to obtain an in vitro 3D-vascularized natural constructs, from scaffolds easily available in nature to use in vivo for the healing of “critical-size” bone defeats. Previously [2] we identified the best preparation methods to obtain suitable SO surface for cell culture. Recently, we have performed a series of in vitro experiments to test the biocompatibility properties of the support; then, cell adhesion tests, viability and proliferation assay were carried out. Further, we tried to induce a vascular-like network organization of Mouse Aortic Endothelial Cells (MAECs) directly on the SOs surface, stimulating the cells with a known angiogenic factor, the Vascular Endothelial Growth Factor (VEGF), getting encouraging preliminary results.


2016 - Bone texture modifications during bone regeneration and osteocyte cell-signaling changes in response to treatment with Teriparatide [Abstract in Rivista]
Smargiassi, Alberto; Checchi, Marta; Cavani, Francesco; Ferretti, Marzia; Palumbo, Carla
abstract

Bone texture modifications during bone regeneration and osteocyte cell-signaling changes in response to treatment with Teriparatide


2016 - COMPARATIVE MORPHOLOGICAL STUDY OF BONE REGENERATION IN DIFFERENT RABBIT CRANIAL OSTEOMOMIES: TRADITIONAL VERSUS NEW GENERATION OSTEOTOMES [Abstract in Rivista]
Ferretti, Marzia; Cavani, Francesco; Checchi, Marta; Smargiassi, Alberto; Anesi, Alexandre; Salvatori, Roberta; Chiarini, Luigi; Palumbo, Carla
abstract

COMPARATIVE MORPHOLOGICAL STUDY OF BONE REGENERATION IN DIFFERENT RABBIT CRANIAL OSTEOMOMIES: TRADITIONAL VERSUS NEW GENERATION OSTEOTOMES


2016 - PRELIMINARY OBSERVATIONS ON SCLERAL OSSICLES IN PERFORMING FUNCTIONALIZED 3D VASCULARIZED SCAFFOLDS FOR "CRITICAL_SIZE" BONE DEFECT HEALING [Abstract in Rivista]
Checchi, Marta; Smargiassi, Alberto; Ferretti, Marzia; Sena, Paola; Benincasa, Marta; Cavani, Francesco; Sola, Marco; Ranieri, Antonio; Stefania, Mitola; Palumbo, Carla
abstract

PRELIMINARY OBSERVATIONS ON SCLERAL OSSICLES IN PERFORMING FUNCTIONALIZED 3D VASCULARIZED SCAFFOLDS FOR "CRITICAL_SIZE" BONE DEFECT HEALING


2016 - The Proteasome Inhibitor Bortezomib Maintains Osteocyte Viability in Multiple Myeloma Patients by Reducing Both Apoptosis and Autophagy: A New Function for Proteasome Inhibitors [Articolo su rivista]
Toscani, Denise; Palumbo, Carla; Dalla Palma, Benedetta; Ferretti, Marzia; Bolzoni, Marina; Marchica, Valentina; Sena, Paola; Martella, Eugenia; Mancini, Cristina; Ferri, Valentina; Costa, Federica; Accardi, Fabrizio; Craviotto, Luisa; Aversa, Franco; Giuliani, Nicola
abstract

Multiple myeloma (MM) is characterized by severely imbalanced bone remodeling. In this study, we investigated the potential effect of proteasome inhibitors (PIs), a class of drugs known to stimulate bone formation, on the mechanisms involved in osteocyte death induced byMMcells. First, we performed a histological analysis of osteocyte viability on bone biopsies on a cohort of 37MMpatients with symptomatic disease. A significantly higher number of viable osteocytes was detected in patients treated with a bortezomib (BOR)-based regimen compared with those treated without BOR. Interestingly, both osteocyte autophagy and apoptosis were affected in vivo by BOR treatment. Thereafter, we checked the in vitro effect of BOR to understand the mechanisms whereby BOR maintains osteocyte viability in bone fromMM patients. We found that osteocyte and preosteocyte autophagic death was triggered during coculturing with MM cells. Our evaluation was conducted by analyzing either autophagy markers microtubule-associated protein light chain 3 beta (LC3B) and SQSTM1/sequestome 1 (p62) levels, or the cell ultrastructure by transmission electron microscopy. PIs were found to increase the basal levels of LC3 expression in the osteocytes while blunting the myeloma-induced osteocyte death. PIs also reduced the autophagic death of osteocytes induced by high-dose dexamethasone (DEX) and potentiated the anabolic effect of PTH(1-34). Our data identify osteocyte autophagy as a new potential target in MM bone disease and support the use of PIs to maintain osteocyte viability and improve bone integrity in MM patients.


2016 - ULTRASTRUCTURAL ASPECTS OF ARTICULAR CARTILAGE AND SUB-CHONDRAL BONE IN PATIENTS AFFECTED BY POST-TRAUMATIC SHOULDER INSTABILITY [Abstract in Atti di Convegno]
Rebuzzi, Manuela; Paolo, Baudi; Ferretti, Marzia; Campochiaro, Gabriele; Gialdini, Mauro; Corradini, Alessandro; Palumbo, Carla; Catani, Fabio
abstract

ULTRASTRUCTURAL ASPECTS OF ARTICULAR CARTILAGE AND SUB-CHONDRAL BONE IN PATIENTS AFFECTED BY POST-TRAUMATIC SHOULDER INSTABILITY


2015 - Effects of PTH(1-34) during fracture healing after experimental bone drilling in rat femur: novel aspects [Abstract in Rivista]
Smargiassi, Alberto; Ferretti, Marzia; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Palumbo, Carla
abstract

The study concerns the role of PTH(1-34) during bone lesion repair. 3-month-old male Sprague-Dawley rats, in which trans-cortical holes were drilled at femur middiaphysis, were divided in groups with/without Teriparatide administration (40g/ Kg/day), and sacrificed at different times (10, 28, 45 days). In 2002 (1) we demonstrated the occurrence of two successive bone forming processes during both skeletal organogenesis and bone repair, i.e. static (SO) and dynamic (DO) osteogenesis: the former (due to stationary osteoblasts, haphazardly grouped in cords) producing preliminary bad quality trabecular bone, the latter (due to typical polarized osteoblasts organized in ordered movable laminae) producing mechanically valid bone tissue. In brief, the primary function of SO is to provide a rigid scaffold, containing osteocytes (i.e. mechano-sensors), to DO-osteoblastic laminae; therefore, in DO mechanical factors can play a crucial role in transduction of mechanical stresses into biological signals. In the present work, histomorphometric analysis showed that, already after 10 days from drilling, notwithstanding the holes are temporarily filled by the same amount of newly-formed trabecular bone (produced by SO) independently from the treatment, the number of movable osteoblast laminae (typical of DO), covering the trabecular surface, is statistically higher in animals submitted to PTH(1-34) administration than in the control ones; this suggests that the mere effect of Teriparatide is to anticipate the occurrence of dynamic osteogenesis involved in the production of good quality bone more suitable to loading. These findings are also supported by the higher values of microhardness as well as the more ordered-fibered texture (observed by polarized light) in treated animals with respect to control ones that strongly indicates the qualitative (instead of quantitative) effect of PTH (1-34) in improving bone healing. The present investigation could be of crucial importance in further translational clinical research in humans to define the best therapeutic strategies in recovering skeletal lesions, particularly in terms of time of administration of PTH(1-34).


2015 - Mineral and Skeletal Homeostasis Influence the Manner of Bone Loss in Metabolic Osteoporosis due to Calcium-Deprived Diet in Different Sites of Rat Vertebra and Femur [Articolo su rivista]
Ferretti, Marzia; Cavani, Francesco; Smargiassi, Alberto; Roli, Laura; Palumbo, Carla
abstract

Rats fed calcium-deprived diet develop osteoporosis due to enhanced bone resorption, secondary to parathyroid overactivity resulting from nutritional hypocalcemia.Therefore, rats provide a good experimental animal model for studying bone modelling alterations during biochemical osteoporosis.Three-month-old Sprague-Dawley male rats were divided into 4 groups: (1) baseline, (2) normal diet for 4 weeks, (3) calcium-deprived diet for 4 weeks, and (4) calcium-deprived diet for 4 weeks and concomitant administration of PTH (1-34) 40 g/Kg/day. Histomorphometrical analyses were made on cortical and trabecular bone of lumbar vertebral body as well as of mid-diaphysis and distal metaphysis of femur. In all rats fed calcium-deprived diet, despite the reduction of trabecular number (due to themaintenance of mineral homeostasis), an intense activity of bone deposition occurs on the surface of the few remaining trabeculae (in answering to mechanical stresses and, consequently, to maintain the skeletal homeostasis). Different responses were detected in different sites of cortical bone, depending on their main function in answering mineral or skeletal homeostasis. This study represents the starting point for work-in-progress researches, with the aim of defining in detail timing and manners of evolution and recovery of biochemical osteoporosis.


2015 - Ultrastructural aspects of articular cartilage and subchondral bone in patients affected by post-traumatic shoulder instability: preliminary observations [Abstract in Rivista]
Baudi, Paolo; Campochiaro, Gabriele; Rebuzzi, Manuela; Ferretti, Marzia; Serafini, Fabio; Catani, Fabio; Palumbo, Carla
abstract

Post traumatic shoulder instability is a frequent condition in young active population. Notwithstanding a lot of data have been collected on capsular-legament lesions and gleno-humeral defects, no data are available on early ultrastructural ostheo-condral damages that are known to be highly associated with the onset of invalidating pathologies, like osteoarthritis (OA). Thus, the mechanisms of joint instability and the identification of which components in the articular complex are primarily affected in instability are of clinical significance, particularly in the light of deepening knowledge on the onset/development of OA. In the present study, biopsies of the articular cartilage and sub-chondral bone were taken from 10 patients (aged 26-40) underwent surgery in Policlinico of Modena. The withdrawals were immediately fixed and embedded for Transmission Electron Microscopy (TEM). The observations were performed in tangential, arcuate, and radial layers of the articular cartilage as well as in sub-chondral bone. TEM observations showed that chondrocytes in the superficial layers (i.e. tangential and arcuate) display normal and very well preserved ultrastructure, probably due to synovial liquid supply; otherwise, chondrocytes in the radial layer (not only in calcified but also in the un-calcified one) show various degrees of degeneration, with cytoplasm partially coerced and variously-sized vacuoles, both signs of suffering; occasionally, in the radial layer, chondrocytes with morphological signs of apoptosis or autophagy were also observed. As far as sub-chondral bone is concerned, osteocytes next the deeper calcified cartilage (within 80-100 micra from the cement line) also show evidences of degeneration, while osteocytes more distant from the osteo-chondral border display normal ultrastructure probably due to the vascular bone supply. In all patients of the study, the ultrastructural features of osteo-chondral complex are not depending on age. The present study represents the first ultrastructural investigation of the articular osteo-chondral complex in shoulder instability, evaluating the state of preservation/viability of both chondrocytes and osteocytes throughout the successive layers of the articular cartilage and sub-chondral bone. These preliminary observations are the basis to understand if the early surgical treatment in shoulder instability could avoid the onset of OA.


2014 - Effect of PTH (1-34) on trabecular bone of rat vertebral body in induced-biochemical osteoporosis by calcium- deprived diet [Abstract in Rivista]
Ferretti, Marzia; Cavani, Francesco; Smargiassi, Alberto; Sena, Paola; Benincasa, Marta; Palumbo, Carla
abstract

Rats fed calcium-deprived diet were used as experimental model for studying bone modelling alterations during biochemical osteoporosis and recovery of bone loss. Such model is suitable to evaluate the possible effects exerted by PTH(1-34) in preventing as well as in recovering metabolic osteoporosis. Three-month-old Sprague Dawley male rats were divided in different groups: some fed normal diet or calcium-deprived diet with/without 40µg/Kg/day PTH(1-34), provided by Eli Lilly-USA, for 4 weeks and some with restoration of normal diet with/without PTH (1-34) for further 4 weeks. To evaluate the occurrence of osteogenesis during the first 4 weeks of the experimental period, rats received three labels of bone deposition at 1st, 20th and 27th day (and then were sacrificed); during the successive 4 weeks (in which those rats previously fed with calcium-deprived diet had restoration of normal diet), animals received three labels of bone deposition at 1st, 7th and 14th day. Histomorphometrical analyses were performed on cortical and trabecular bone taken from the central level of the 5th lumbar vertebral body, transversely sectioned. The results showed that differences among the groups were observed mainly in trabecular bone with respect to cortical one, thus underlining the different role of the two types of bone architecture in mineral and skeletal homeostasis. Concerning trabecular bone, the observations showed that administration of PTH (1-34) during calcium-deprived diet and/or during the restoration of normal diet induces higher deposition of trabecular bone with respect to that recorded in rats that never received PTH(1-34), neither during calcium-deprived diet nor during restoration of normal diet. Since increments of trabecular bone are detectable only after the period of diet restoration (but not before), the authors suggest that a chronic administration of PTH (1-34) is necessary to achieve appreciable results on bone mass recovery.


2014 - Ferutinin dose-dependent effects on uterus and mammary gland in ovariectomized rats [Articolo su rivista]
Ferretti, Marzia; Cavani, Francesco; Manni, Paola; Carnevale, Gianluca; Bertoni, Laura; Zavatti, Manuela; Palumbo, Carla
abstract

The present paper completes our recent study on the effects of phytoestrogen ferutinin in preventing osteoporosis and demonstrating the superior osteoprotective effect of a 2 mg/kg/day dose in ovariectomized (OVX) rats, compared to both estrogens and lower (0.5, 1 mg/kg/day) ferutinin doses. Morphological and morphometrical analyses were performed on the effects of different doses of ferutinin administrated for one month on uterus and on mammary gland of Sprague-Dawley OVX rats, evaluated in comparison with the results for estradiol benzoate. To verify whether ferutinin provides protection against uterine and breast cancer, estimations were made of both the amount of cell proliferation (by Ki-67), and the occurrence of apoptosis (by TUNEL), two processes that in unbalanced ratio form the basis for cancer onset. The results suggest that the effects of ferutinin are dose dependent and that a 2 mg/kg/day dose might offer a better protective action against the onset of both breast and uterine carcinoma compared to ferutinin in lower doses or estradiol benzoate, increasing cellular apoptosis in glandular epithelia.


2014 - Oral surgery biomaterials: analyses of Al2O3-treated titanium surfaces tested with fibroblast and osteocyte cell lines [Abstract in Rivista]
Smargiassi, Alberto; Ferretti, Marzia; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Zaffe, Davide; Facciani, Valentino; Gabrel, Ivano; Palumbo, Carla
abstract

Two different cell lines - MLO-Y4 (murine osteocytes) and 293 (human fibroblasts) - cultured for 48 hours in standard media were used to analyse engineered bio-materials (i.e. Al2O3 shot-peened titanium surfaces). Distribution, density and expression of adhesion molecules (fibronectin and vitronectin) were evaluated under scanning electron microscope (SEM) and confocal microscope (CM) as previously described [1]. The engineered biomaterial surfaces showed under SEM irregular morphology displaying variously-shaped spicules, obtained by shooting different-in-size particles of Al2O3 against the scaffolds of biomaterial. DAPI and fluorochrome-conjugated antibodies were used to highlight nuclei, fibronectin and vitronectin, under CM; cell distribution was analysed after Gold-Palladium sputtering of samples by SEM. Both SEM and CM observations showed better outcome in terms of cell adhesion and distribution in treated titanium surfaces with respect to the untreated ones. The results obtained clearly showed that this kind of surface-treated titanium, used to manufacture devices for dental implantology: i) is very suitable for cell colonization, essential prerequisite for the best osseointegration, and ii) represents an excellent solution for the development of further engineered implants with the target to obtain recovery of dental function stable over time. Further studies on these Al2O3 shot-peened-titanium surfaces, both in vitro and in vivo, will be needed to obtain accurate definition of better biomaterial outcome, also after additional treatments. References [1] Palumbo et al. (2013) Immunocytochemical and structural comparative study of committed versus multipotent stem cells cultured with different biomaterials. Micron 47: 1–9.


2014 - PRELIMINARY FINDINGS OF A POTENZIATED PIEZOSURGERGICAL DEVICE AT THE RABBIT SKULL [Abstract in Atti di Convegno]
Anesi, Alexandre; Palumbo, Carla; Ferretti, Marzia; Salvatori, Roberta; Cavani, Francesco; Chiarini, Luigi
abstract

The number of available ultrasonic osteotomes has remarkably increased. In vitro and in vivo studies have revealed differences between conventional osteotomes, such as rotating or sawing devices, and ultrasound-supported osteotomes (Piezosurgery®) regarding the micromorphology and roughness values of osteotomized bone surfaces. Objective: the present study compares the micro-morphologies and roughness values of osteotomized bone surfaces after the application of rotating and sawing devices, Piezosurgery Medical® and Piezosurgery Medical New Generation Powerful Handpiece. Methods: Fresh, standard-sized bony samples were taken from a rabbit skull using the following osteotomes: rotating and sawing devices, Piezosurgery Medical® and a Piezosurgery Medical New Generation Powerful Handpiece. The required duration of time for each osteotomy was recorded. Micromorphologies and roughness values to characterize the bone surfaces following the different osteotomy methods were described. The prepared surfaces were examined via light microscopy, environmental surface electron microscopy (ESEM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM) and atomic force microscopy. The selective cutting of mineralized tissues while preserving adjacent soft tissue (dura mater and nervous tissue) was studied. Bone necrosis of the osteotomy sites and the vitality of the osteocytes near the sectional plane were investigated, as well as the proportion of apoptosis or cell degeneration. Results and Conclusions: The potential positive effects on bone healing and reossification associated with different devices were evaluated and the comparative analysis among the different devices used was performed, in order to determine the best osteotomes to be employed during cranio-facial surgery.


2014 - PROTEASOME INHIBITORS MODULATE OSTEOCYTE DEATH AND AUTOPHAGY IN MULTIPLE MYELOMA. [Abstract in Rivista]
Toscani, Denise; Palumbo, Carla; Ciullo, Alessandra; Ferretti, Marzia; Bolzoni, Marina; Guasco, Daniela; Palma, Benedetta Dalla; Aversa, Franco; Giuliani, Nicola
abstract

Background: Cell death and autophagy are the main cellular processes involved in the regulation of bone remodeling by osteocytes. Recently we have demonstrated that an increased osteocyte death is involved in multiple myeloma (MM)-induced osteolysis through the upregulation of osteoclast recruitment. Aims: Because proteasome inhibitors including Bortezomib (BOR) are known to be able to target osteoblasts in this study we have investigated the potential effect of these drugs on osteocytes and their cell death and autophagy. Methods: Firstly the effect of the proteasome inhibitors BOR and MG262 on osteocyte viability was evaluated in vitro in murine osteocytic cell line MLO-Y4 and in the human pre-osteocytic one HOB-01. Both cell lines were co-coltured for 48 hours in the presence or absence of the human myeloma cell lines (HMCLs) RPMI8226 and JJN3, placed in a transwell insert in the presence or the absence of BOR or MG262. Moreover the effect of proteasome inhibitors on dexamethasone (DEX)-induced MLO-Y4 death, obtained at high doses (10-5-10-6M), was checked in combination with PTH(1-34). To evaluate the presence of autophagy and apoptosis in osteocytes, we checked the expression of both autophagic marker LC3 and apoptotic marker APAF-1 by confocal microscopy in the co-colture system with MLO-Y4 and RPMI-8226. Finally we performed a retrospective histological evaluation on bone biopsies of a cohort of 31 newly diagnosis MM underwent to different treatments including BOR-based regimen. Bone biopsies were obtained at the diagnosis and after an average time of 12 months of treatment. Osteocyte viability was evaluated in a total of 500 lacunae per histological sections. Results: The in vitro treatment with BOR or MG262 significantly blunted MLO-Y4 and HOB-01 cell death. Similarly, DEXinduced MLO-Y4 death was reduced by proteasome inhibitors. Interestingly, we found that both proteasome inhibitors potentiated the PTH (1-34) short-term effects on DEX-induced osteocyte death. Prevalence of autophagic cell death compared to apoptosis was observed in this system. In line with these data, we showed that neither the HMCLs nor treatment with DEX increase the apoptotic death and caspase 3 activation in both MLO-Y4 and HOB-01 cell lines. BOR treatment increased the basal level of LC3 indicating a pro-survival and protective function of autophagy against the BOR-induce stress. On the contrary, when the cells undergo to a stronger stress such as in the presence of HMCLs or by treatment with high dose of DEX we found that both proteasome inhibitors blocked autophagic cell death in osteocytes. In the in vivo study we found a significant increase of the number of viable osteocytes in MM patients treated with BOR-based regimen as compared to those treated without BOR (% median increase: +6% vs. +1.30%; p=0.017). Patients treated with BOR alone showed the highest increase of osteocyte viability, as compared to those either treated without BOR (+11.6% vs. +1.3%, p=0.0019) or treated with BOR plus DEX (+11.6% vs. +4.4%, p=0.01). On the other hand, any significant difference was not observed in patients treated with Thalidomide (THAL) or Immunomodulatory drugs (IMiDs) than in those untreated with these drugs (p= 0.7). A multiple regression non-parametric analysis showed that BOR had a significant positive impact on osteocyte viability (p=0.042) whereas THAL/IMiDs as well as Zoledronic acid (ZOL) treatments have not (p=0.2). BOR also counterbalanced the negative effect of DEX treatment (p=0.035). Summary/Conclusion: Our data suggest that proteasome inhibitors blunted osteocyte cell death induced by MM cells and DEX through the modulation of the autophagy and potentiated the effect of PTH. Overall our in vitro and in vivo data support the use of BOR to improve bone integrity in MM patients.


2013 - Chondrocyte expression of apoptotic and pro-inflammatory factors in the development of post- traumatic arthritis in humans [Abstract in Rivista]
Sena, Paola; Benincasa, Marta; Cavani, Francesco; Ferretti, Marzia; Smargiassi, Alberto; Manfredini, Giuseppe; Palumbo, Carla
abstract

The development of post-traumatic arthritis following intra-articular fracture remains an important unsolved clinical problem. The possibility that extensive chondrocyte apoptosis occurs following intra-articular fracture, thus contributing to the development of post-traumatic arthritis, has received increasing attention [1]. It has been demonstrated the existence of a direct correlation between the rate of apoptosis and the severity of osteoarthritis [2]. Pharmacologic inhibitors of enzymes involved in apoptosis have been explored as potential therapeutic agents [3]. In the present study we aimed to deepen the characterization of apoptotic mediators, expressed by chondrocytes, involved in human post-traumatic arthritis following intra-articular fracture and the possible implication of pro-inflammatory receptors in arthritis. The expression of a panel of pro/anti apoptotic factors (Caspase-3, PARP-1, BCL2) and inflammation-related receptors (ChemR23) were analysed in chondrocytes from patients undergoing surgery for intra-articular calcaneal fractures. The factors were investigated by immunofluorescence coupled with confocal analysis and western blotting, followed by densitometric evaluation of chondrocyte cultures harvested from patients with intra-articular fractures compared with control ones. The results clearly demonstrated that a statistically significant difference exists in the expression of pro/anti apoptotic factors and ChemR23 between fractured and control patients. In conclusion our data suggest that increased chondrocyte death, occurring after cartilage injury together with inflammatory process, could play a pivotal role in the onset of arthritic disease. References [1]. Hembree W.C. et al. (2007) Viability and apoptosis of human chondrocytes in osteochon-dral fragments following joint trauma. J Bone Joint Surg Br 89(10): 1388-95. [2] Kim H.A. et al. (2000) Apoptotic chondrocyte death in human osteoarthritis. J Rheumatol 27: 455–462. [3] D'Lima D. et al. (2006) Caspase inhibitors reduce severity of cartilage lesions in experi-mental osteoarthritis. Arthritis Rheum 54(6): 1814-1821.


2013 - Immunocytochemical and structural comparative study of committed versus multipotent stem cells cultured with different biomaterials. [Articolo su rivista]
Palumbo, Carla; Baldini, Andrea; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Ferretti, Marzia; Zaffe, Davide
abstract

The aim of this work was the comparison of the behavior of committed (human osteoblast cells - hOB - from bone biopsies) versus multipotent (human dental pulp stem cells - hDPSC - from extracted teeth) cells, cultured on shot-peened titanium surfaces, since the kind of cell model considered has been shown to be relevant in techniques widely used in studies on composition/morphology of biomaterial surfaces. The titanium surface morphology, with different roughness, and the behavior of cells were analyzed by confocal microscope (CM), scanning electron microscope (SEM) and X-ray microanalysis. The best results, in terms of hOB adhesion/distribution, were highlighted by both CM and SEM in cultured plates having 20-mum-depth cavities. On the contrary, CM and SEM results highlighted the hDPSC growth regardless the different surface morphology, arranged in overlapped layers due to their high proliferation rate, showing their unfitness in biomaterial surface test. Nevertheless, hDPSC cultured inside 3D-matrices reproduced an osteocyte-like three-dimensional network, potentially useful in the repair of critical size bone defects. The behavior of the two cell models suggests a different use in biomaterial cell cultures: committed osteoblast cells could be appropriate in selecting the best surfaces to improve osseointegration, while multipotent cells could be suitable to obtain in vitro osteocyte-like network for regenerative medicine. The originality of the present work consists in studying for the first time two different cell models (committed versus multipotent) compared in parallel different biomaterial cultures, thus suggesting distinct targets for each cellular model. Copyright 2012 Elsevier Ltd. All rights reserved.


2013 - Induced Biochemical osteoporosis: Effects of 1-month calcium–deprived diet on rat bone remodelling with/without contemporary administration of PTH(1-34) [Abstract in Rivista]
Ferretti, Marzia; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Smargiassi, Alberto; Palumbo, Carla
abstract

It is known that rats fed calcium-deprived diet develop osteoporosis due to en-hanced bone resorption secondary to parathyroid overactivity resulting from nutritional hypocalcemia. Therefore, rats provide a good experimental animal model for studying bone remodelling alterations during biochemical osteoporosis. This preliminary study is performed in 3 month-old Sprague Dawley male rats, divided into 4 groups (5 rats each): 1) base line, 2) normal diet for 4 weeks, 3) calcium-deprived diet for 4 weeks; 4) calcium-deprived diet for 4 weeks plus contemporary administration of PTH(1-34) 40µg/kg/day. Three labels of osteogenesis were performed at 1st , 20th and 27th day of experimental period in order to evaluate bone formation during animal treatment. His-tomorphometrical analyses were performed on cortical bone of femoral diaphyses, as well as on trabecular bone of distal femoral metaphyses, both transversely sectioned. The preliminary results showed that at femur mid-diaphyseal level the diet induced a reduction of cortical bone area (even if not significant) with enlargement of the medul-lary canal due to endosteal resorption, while periosteal neo-deposition is similar in all groups and particularly abundant in those periosteal regions mainly devoted in answering the mechanical demands. PTH(1-34) treatment seems to reduce endosteal resorption only in those surfaces where periosteal mechanical loading are less consistent. Conversely, PTH(1-34) treatment doesn't seem to affect osteoblast activity. Moreover, in distal femoral metaphyses, diet induced osteoclast activity, with a decrease in the amount of trabecular bone volume, confirming that this architecture is mainly devoted in answering the metabolic demands. The novelty of the proposed model Is the contemporary administration of PTH(1-34) together with calcium deprived diet to evaluate induced-biochemical osteoporosis. This model seems a good starting point for successive studies in order to study bone alterations during unbalanced calcium metabolism frequently occurring in aging and to define time and manner of bone mass recovery.


2013 - Myeloma-Induced Osteocyte Death Was Blunted By Proteasome Inhibitors Through The Modulation Of Autophagy [Poster]
Toscani, Denise; Palumbo, Carla; Palma, Benedetta Dalla; Bolzon, Marina; Ferretti, Marzia; Sena, Paola; Guasco, Daniela; Martella, Eugenia; Aversa, Franco; Giuliani, Nicola
abstract

Osteocytes are critical in the maintenance of bone integrity regulating bone remodeling through the cell death and autophagy, a cellular process stress-induced to prolong cell survival but when induced excessively can cause cell death. Recently we have demonstrated that an increased osteocyte death is involved in multiple myeloma (MM)-induced osteolysis. However the mechanisms involved in this process as well as the effect of the proteasome inhibitors able to stimulate bone formation are not known and have been investigated in this study. Firstly the effect of the proteasome inhibitors BOR and MG262 on osteocyte viability was evaluated in vitro in murine osteocytic cell line MLO-Y4 and in the human pre-osteocytic one HOB-01. Both cell lines were co-coltured for 48 hours in the presence or absence of the human myeloma cell lines (HMCLs) RPMI8226 and JJN3, placed in a traswell insert. The treatment for 12-24 hours with (BOR) (2nM) and MG262 (10nM) significantly blunted MLO-Y4 and HOB-01 cell death. In addition, dexamethasone (DEX)-induced MLO-Y4 apoptosis, obtained at high doses (10-5-10-6 M), was reduced by the treatment with proteasome inhibitors. Interestingly, we found that PTH short-term treatment potentiated the in vitro effects of proteasome inhibitors on DEX-induced osteocyte death. To evaluate the presence of autophagy in osteocytes, we checked the expression of the autophagic marker LC3 both by confocal microscopy and western blot analysis in the co-colture system with MLO-Y4 and RPMI-8226. Prevalence of autophagic cell death and in a lesser extent apoptosis was observed in this system. BOR increased the basal level of LC3 indicating a pro-survival and protective function of autophagy against the BOR-induce stress. On the contrary, when cells undergo to a stronger stress such as in the presence of HMCLs or by treatment with high dose of DEX we found that both proteasome inhibitors BOR and MG262 blocked autophagic cell death in osteocytes. To translate our in vitro evidence in a clinical perspective, thereafter we performed a histological evaluation on bone biopsies of a cohort of 37 newly diagnosis MM patients 31 of them with symptomatic MM and 6 with smoldering MM (SMM). The 55% of patients with MM have evidence of osteolytic lesions at the X-rays survey. Bone biopsies were obtained at the diagnosis and after an average time of 12 months of treatment or observation. Osteocyte viability was evaluated in a total of 500 lacunae per histological sections. A significant increase of the number of viable osteocytes was demonstrated in MM patients treated with BOR-based regimen as compared to those treated without BOR (% median increase: +6% vs. +1.30%; p=0.017). Patients treated with BOR alone showed the highest increase of osteocyte viability, as compared to those either treated without BOR (+11.6% vs. +1.3%, p=0.0019) or treated with BOR plus DEX (+11.6% vs. +4.4%, p=0.01). A reduction of both osteocyte apoptosis and autophagy was demonstrated by TUNEL assays and confocal microscopy. On the other hand, any significant difference was not observed in patients treated with Thalidomide (THAL) or Immunomodulatory drugs (IMiDs) than in those untreated with these drugs (p= 0.7). A multiple regression non-parametric analysis showed that BOR had a significant positive impact on osteocyte viability (p=0.042) whereas THAL/IMiDs as well as Zoledronic acid (ZOL) treatments have not (p=0.2). BOR also counterbalanced the negative effect of DEX treatment (p=0.035). Our data suggest that proteasome inhibitors blunted osteocyte cell death induced by MM cells and DEX through the modulation of the autophagy supporting their use to improve bone integrity in MM patients.


2013 - The problem of bone lamellation: An attempt to explain different proposed models [Articolo su rivista]
Marotti, Gastone; Ferretti, Marzia; Palumbo, Carla
abstract

Collagen texture and osteocyte distribution were analyzed in human woven- and lamellar-bone using scanning and transmission electron microscopy. We provide data substantiating the concept that lamellar bone is made up of an alternation of dense-acellular lamellae and loose-cellular lamellae, all exhibiting an interwoven texture of collagen fibers. An attempt is also made to explain how the present findings might conform to those of authors whose models propose orderly, geometric arrangements of collagen fibers inside bony lamellae. Such a comparison is possible because the present investigation analyzes split loose lamellae and tangentially-sectioned dense lamellae. It emerged that only loose lamellae can be dissected, revealing a loose interwoven collagen texture and halved osteocyte lacunae. Dense lamellae cannot be split because of their compactness. The analysis of tangentially sectioned dense lamellae demonstrates that they consist of a network of interwoven collagen fiber bundles. Inside each bundle, collagen fibers run parallel to each other but change direction where they enter adjacent bundles, at angles as described by other authors whose TEM investigations were performed at a much higher magnification than those of the present study. Consequently, what these authors consider to be a lamella are, instead, bundles of collagen fibers inside a lamella. There is discussion of the role played by the manner of osteocyterecruitment in the deposition of lamellar- and wovenbone and how the presence of these cells is crucial for collagen spatial arrangement in bone tissues.


2012 - APPLICATION OF POLY-L-LACTIDE SCREWS IN FLAT FOOT SURGERY: HISTOLOGICAL AND RADIOLOGICAL ASPECTS OF BIO-ABSORPTION OF DEGRADABLE DEVICES. [Articolo su rivista]
Sena, Paola; Manfredini, G.; Barbieri, Claudia; Mariani, Francesco; Tosi, Giovanni; Ruozi, Barbara; Ferretti, Marzia; Marzona, Laura; Palumbo, Carla
abstract

The flat foot in childhood is a condition frequently observed in orthopedic practice but it is still debated when and in which patients surgical corrective treatment is appropriate; recently, the application of poly-L-lactic-acid (PLLA) screws was proposed. The present study investigates a group of 33 patients treated with PLLA expansion endorthesis in order to evaluate the deformity correction. Clinical and radiological outcomes in patients were correlated with: a) morphological characterization of screws both before and after being removed from patients, when necessary; b) histological and bio-molecular evaluation of degradation processes of the implants, focusing attention on the correlation between the cellular cohort involved in inflammatory reaction and the bio-absorption degree of PLLA screws. Deformity correction was mostly achieved, with minimal need of screw removal; the results obtained clearly show the occurrence of chronic rather than acute inflammation in removed screw specimens.At the histological level, after biomaterial implantation, the sequence of events occurring in the surrounding tissues ultimately ends in the formation of foreign body giant cells (FBGCs) at the tissue/material interface; but the mechanisms which influence the fate of screw implants, i.e. the resolution of acute inflammation rather than the progression towards chronic inflammation, are of crucial importance for biodegradable materials like “polylactic acid”. In fact, the FBGC response ensures a long-term mechanism which eliminates the foreign material from the body, but at the same time the implications of prolonged FBGC responses, which generate negative side effects, could significantly impede the healing progress.


2012 - Effects of different doses of ferutinin on bone formation/resorption in ovariectomized rats. [Articolo su rivista]
Cavani, Francesco; Ferretti, Marzia; Carnevale, Gianluca; Bertoni, Laura; Zavatti, Manuela; Palumbo, Carla
abstract

This study analyzes the effects of different doses of ferutinin on bone loss caused by estrogen deficiency in ovariectomized rats, in comparison with estradiol benzoate. Thirty female Sprague-Dawley rats were ovariectomized and treated for 30 days from the day after ovariectomy. Static/dynamic histomorphometric analyses were performed on trabecular and cortical bone of lumbar vertebrae and femurs. Very low weight increments were recorded only in all F-OVX groups, with respect to the others. Although the great differences in weight, that could imply a decrease of bone mass in F-OVX groups compared to the control ovariectomized group (C-OVX), trabecular bone in lumbar vertebrae did not show significant differences, suggesting that ferutinin, opposing estrogen deficiency, inhibits bone resorption. Newly formed cortical bone was always low in all F-OVX groups and high in C-OVX, suggesting that it is mainly devoted in answering mechanical demands. In contrast, in distal femoral metaphyses, trabecular bone was reduced and the number of osteoclasts was increased in C-OVX with respect to all other groups, suggesting that it is mainly devoted in answering metabolic demands; moreover, ferutinin dose of 2 mg/kg seemed to be more effective than the lower doses used and estrogens, particularly in those skeletal regions with higher metabolic activity. Our results suggest that the role of ferutinin in preventing osteoporosis caused by estrogen deficiency is expressed in decreasing bone erosion; moreover, in all F-OVX groups bone turnover is very low and seems correlated to the trivial body weight increase, which, in turn, depends on ferutinin treatment.


2012 - Increased osteocyte death in multiple myeloma patients: role in myeloma-induced osteoclast formation [Articolo su rivista]
Giuliani, N; Ferretti, Marzia; Bolzoni, M; Storti, P; Lazzaretti, M; DALLA PALMA, B; Bonomini, S; Martella, E; Agnelli, L; Neri, A; Ceccarelli, F; Palumbo, Carla
abstract

The involvement of osteocytes in multiple myeloma (MM)-induced osteoclast (OCL) formation and bone lesions is still unknown. Osteocytes regulate bone remodelling at least partially, as a result of their cell death triggering OCL recruitment. In this study, we found that the number of viable osteocytes was significantly smaller in MM patients than in healthy controls, and negatively correlated with the number of OCLs. Moreover, the MM patients with bone lesions had a significantly smaller number of viable osteocytes than those without, partly because of increased apoptosis. These findings were further confirmed by ultrastructural in vitro analyses of human preosteocyte cells cocultured with MM cells, which showed that MM cells increased preosteocyte death and apoptosis. A micro-array analysis showed that MM cells affect the transcriptional profiles of preosteocytes by upregulating the production of osteoclastogenic cytokines such as interleukin (IL)-11, and increasing their pro-osteoclastogenic properties. Finally, the osteocyte expression of IL-11 was higher in the MM patients with than in those without bone lesions. Our data suggest that MM patients are characterized by a reduced number of viable osteocytes related to the presence of bone lesions, and that this is involved in MM-induced OCL formation.


2012 - Osteocyte apoptosis and absence of bone remodeling in human auditory ossicles and scleral ossicles of lower vertebrates: a mere coincidence or linked processes? [Articolo su rivista]
Palumbo, Carla; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Ferretti, Marzia
abstract

Considering the pivotal role as bone mechanosensors ascribed to osteocytes in bone adaptation to mechanical strains, the present study analyzed whether a correlation exists between osteocyte apoptosis and bone remodeling in peculiar bones, such as human auditory ossicles and scleral ossicles of lower vertebrates, which have been shown to undergo substantial osteocyte death and trivial or no bone turnover after cessation of growth. The investigation was performed with a morphological approach under LM (by means of an in situ end-labeling technique) and TEM. The results show that a large amount of osteocyte apoptosis takes place in both auditory and scleral ossicles after they reach their final size. Additionally, no morphological signs of bone remodeling were observed. These facts suggest that (1) bone remodeling is not necessarily triggered by osteocyte death, at least in these ossicles, and (2) bone remodeling does not need to mechanically adapt auditory and scleral ossicles since they appear to be continuously submitted to stereotyped stresses and strains; on the contrary, during the resorption phase, bone remodeling might severely impair the mechanical resistance of extremely small bony segments. Thus, osteocyte apoptosis could represent a programmed process devoted to make stable, when needed, bone structure and mechanical resistance.


2012 - Proteasome Inhibitors Block Myeloma-Induced Osteocyte Death in Vitro and in Vivo in Multiple Myeloma Patients [Abstract in Rivista]
Toscani, Denise; Palma, Benedetta Dalla; Palumbo, Carla; Ferretti, Marzia; Bolzoni, Marina; Guasco, Daniela; Mancini, Cristina; Martella, Eugenia; Lazzaretti, Mirca; Pedrazzoni, Mario; Aversa, Franco; Giuliani, Nicola
abstract

Multiple myeloma (MM) is characterized by a severe unbalanced and uncoupling bone remodeling leading to osteolysis. We have recently shown that osteocytes are involved in MM-induced osteolysis through an increased cell death. Accordingly MM patients are characterized by a reduced number of viable osteocytes related to the presence of bone lesions. Proteasome inhibitors currently used in the treatment of MM are able to stimulate osteoblast formation but their potential effects on osteocyte death are not known and have been investigated in this study both in vitro and in vivo. Osteocytic MLO-Y4 cells or human pre-osteocytic HOB-01 cells were co-cultured for 48 hours in the presence or absence of the human myeloma cell lines (HMCLs) JJN3 or RPMI-8226 placed in a transwell insert. A significantly reduction of ostecyte viability was observed (median percent reduction of MLO-Y4 viability: -16% and -30%, respectively). The treatment for 12–24 hours with Bortezomib (BOR) (2nM) or other proteasome inhibitors such as MG262 (10nM) or MG132 (100nM) significantly blunted MLO-Y4 and HOB-01 cell death. Similarly, Dexamethasone (DEX)-induced MLO-Y4 apoptosis, obtained at pharmacological doses (10–4–10–5 M), was significantly reduced by the treatment with proteasome inhibitors. To translate our in vitro data into a clinical perspective we performed a retrospective histological evaluation on bone biopsies of a cohort of 40 newly diagnosis MM patients (24 male and 16 female, median age: 68 years) 34 of them with symptomatic MM and 6 with smoldering MM (SMM). The 58% of patients with symptomatic MM have evidence of osteolytic lesions at the X-rays survey. Bone biopsies were obtained in both symptomatic MM and SMM at diagnosis and after an average time of 12 months of treatment or observation, respectively. The 68% of patients with symptomatic MM were treated with a BOR-based regimen while 42% do not. Moreover the 58% of MM patients received DEX and the 59% Thalidomide (TAL). Zoledronic acid (ZOL) was infused monthly in the 60% of MM patients. Osteocyte viability was evaluated in a total of 500 lacunae per histological sections, corresponds to the total number of osteocyte lacunae in the bone biopsies. The number of viable osteocytes and the number of degenerated or apoptotic osteocytes and empty lacunae have been evaluated. In patients with SMM no significant change was observed in the number of viable osteocytes in the two histological evaluations carried out (median percent change: +1.2, p=0.68, NS). In symptomatic MM patients the mean percent change of the osteocyte viability was not correlated with the response rate to treatment (R2 0.01, p=NS). A significant increase of the number of viable osteocytes was demonstrated in MM patients treated with BOR-based regimen as compared to those treated without BOR (% median increase of osteocyte viability: +6% vs. +1.30%, Mann-Whitney test: p=0.017). Patients treated with BOR alone showed the highest increase of osteocyte viability that was statistical significant in comparison with that observed either in patients treated without BOR (+11.6% vs. +1.3%, p=0.0019) or in those treated with BOR plus DEX (+11.6% vs. +4.4%, p=0.01). On the contrary, no significant difference was observed in patients treated with TAL than in those treated without TAL (p= 0.7, NS) as well as patients treated with ZOL compared to those untreated showed no significant difference in the number of viable osteocytes (p=0.18, NS). To confirm the role of the different drug treatment on the osteocyte viability we perform a multiple regression non-parametric analysis showing that BOR had a significant positive impact on osteocyte viability (p=0.042) whereas ZOL and TAL have not (p>0.2,NS) and it counterbalanced the negative effect of DEX treatment (p=0.035). In conclusion our in vitro and in vivo data suggest the proteasome inhibitors block osteocyte death induced by MM cells cou


2012 - Role of osteocyte apoptosis in peculiar ossicles of the hearing sense organ: preliminary observations on hearing loss and osteoporosis [Abstract in Rivista]
Palumbo, Carla; Presutti, Livio; Genovese, Elisabetta; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Ferretti, Marzia
abstract

Starting point of the present study is the osteocyte role in bone remodelling that allows bone adaptation to mechanical load [1-3]. Bone remodelling has been investigated in relation to the occurrence of apoptosis [4] to understand if and how the process of programmed cell death interferes with bone turnover. In 1998, in a study on human middle ear, Marotti et al. [5] demonstrated that: 1) over 40% of osteo-cytes are dead within the 2nd year of age (but the authors were not able to demonstrate if osteocyte death occurred by degeneration or apoptosis); 2) bone remodelling occurs only occasionally. Recently [6], we showed that: 1) osteocytes of human auditory ossicles die by apoptosis; 2) also osteocytes located inside scleral ossicles of lower vertebrate eye (reptiles and birds) phylogenetically so far from human auditory ossicles are widely affected by apoptosis (about 60%); 3) in scleral ossicles bone turnover never occur. It is to be noted that both auditory ossicles of human ear and scleral ossicles of vertebrate eye are peculiar bony segments continuously submitted to stereotyped stresses and strains, with specialized func-tions: the first are involved in sound wave transmission and the latter protect the eyeball against deformation during the movement and have a role in visual accomodation, providing attachment for the ciliary muscles. In both cases, bone remodelling might severely impair, by resorption, the mechanical resistance of these extremely small specialized bony segments. Thus, we suggested that in auditory and scleral ossicles, submitted to stereotyped loading for all life, bone mechanical adaptation is not needed and osteocyte programmed death could represent the mechanism to avoid bone remodelling and to make stable, when necessary, bone structure and mechanical resistance. More recently, to confirm this hypothesis, clinical data were collected from a cohort of patients aged 55-85 years affected by hearing loss. The main target of the present study is to exclude any correlation between hearing loss and osteoporosis. During osteoporosis, unbalanced bone turnover causes the bone depletion in skeletal segments; such condition, in the peculiar ossicles of human middle ear, should imply hearing impairment. Our preliminary observations indicate, instead, that osteoporotic patients do not show higher percentage of hearing loss with respect to non osteoporotic ones. This evidence is ascribable to osteocyte apoptosis of auditory ossicles that avoid bone remodelling, thus assuring the integrity of such bony segments also in osteoporotic conditions. References [1] Turner (1991) Omeostatic control of bone structure: an application of feed-bach theory. Bone 12: 203-217. [2] Turner and Forwood (1995) What role does the osteocyte network play in bone adaptation? Bone 16: 283-285. [3] Marotti (1996) The structure of bone tissue and the cellular control oftheir deposition. IJAE 101(4): 26-79. [4] Noble et al. (1997) Identification of apoptotic changes in osteocytes in normal and pathological human bone. Bone 20: 273-282. [5] Marotti et al. (1998) Morphometric investigation on osteocytes in human auditory ossicles. Ann Anat 180: 449-453. [6] Palumbo et al. (2012) Osteocyte apoptosis in human auditory ossicles and scleral ossicles of lower ver-tebrates: a mere coincidence or linked processes? Calcif. Tissue Int. 90: 211-218.


2012 - ROLE OF PHYTOESTROGEN FERUTININ IN PREVENTING/RECOVERING BONE LOSS: RESULTS FROM EXPERIMENTAL OVARIECTOMIZED RAT MODEL [Capitolo/Saggio]
Palumbo, Carla; Cavani, Francesco; Bertoni, Laura; Ferretti, Marzia
abstract

In the Chapter 35 of the book are reported observations of recent pubblications on the effect of ferutinin in preventing/recovering severe osteoporosis secondary to ovariectomy in rats. On the basis of the results so far obtained, the authors suggest to enumerate ferutinin among the osteoprotective substances. This fact acquires a more relevant importance in the light of recent tenable evidences reported from various authors concerning the absence of negative side effects by some phytoestrogens (particularly genistein, 8-prenylnaringenin, reveratrol and red clover extract) on the tropism of various organs commonly targeted by estrogens. In conclusion, the results reported not only provide evidence that ferutinin can significantly prevent/recover ovariectomy-induced bone loss in rats, but also that it could protect against the onset of uterus cancer. Although the putative undesired estrogenic-like side effects on uterus of such phytoestrogen have not yet been fully investigated, ferutinin could be an interesting safer alternative new candidate for HRT in treatment of post-menopausal symptoms, since it seems to protect from bone loss induced by ovariectomy (Palumbo et al., 2009; Ferretti et al., 2010) and in part to mime the ovarian endocrine function during menopause.


2012 - Striated muscle fiber apoptosis after experimental tendon lesion in a rat model [Articolo su rivista]
Palumbo, Carla; Rovesta, Claudio; Ferretti, Marzia
abstract

Tendon lesions induce muscular atrophy, the nature of which has not yet been clearly related to lesion etiology and entity. In the present study, tendon and muscle alterations were assessed after experimental tendon lesion of the Infraspinatus muscle in young rats. The consequences of lesions differed on the basis of both extension and injured tissue vascularization, that is apoptosis and/or degeneration, differing mainly by energy demands: apoptosis requires high energy levels (proportional to vascular supply), but degeneration does not. It is well known that tendons are poorly supplied with blood compared with muscular masses, which are abundantly vascularized. Five weeks after tendon surgical section, tendon/muscle samples were taken for TUNEL and transmission electron microscopy. The structural results reported here identified different tendon/muscle alterations: degeneration of tendon without signs of apoptosis, and atrophy of muscle fibers due only to apoptosis. This led to the formulation of the following hypothetical sequence of events: a tendon lesion, not recovering quickly due to the poor tendon blood supply, results in degeneration of the injured tendon, which, in turn, induces a partial disuse of the muscle mass, which consequently atrophies (proportionally to the severity of tendon lesion) by striated muscular fiber apoptosis. The authors suggest that the different behavior of the two tissues depends on the marked difference in their vascularization.


2012 - Structural and histomorphometric evaluations of ferutinin effects on the uterus of ovariectomized rats during osteoporosis treatment [Articolo su rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Benincasa, Marta; Sena, Paola; Carnevale, Gianluca; Zavatti, Manuela; Vittoria Di, Viesti; Zanoli, Paola; Palumbo, Carla
abstract

Aims: The effects of chronic administration of Ferutinin (phytoestrogen found in the plants of genus Ferula),compared with those elicited by estradiol benzoate, were evaluated, following ovariectomy, on the uterus ofovariectomized rats as regard weight, size, structure and histomorphometry.Main methods: The experimental study included 40 female Sprague–Dawley rats, assigned to two different protocols,i.e. preventive and recovering. In the preventive protocol, ferutinin (2 mg/kg/day)was orally administeredfor 30 days, starting from the day after ovariectomy; in the recovering protocol, ferutinin was administered, atthe same dosage, for 30 days starting fromthe 60th day after ovariectomy, when osteoporosiswas clearly established.Its effects were compared with those of estradiol benzoate (1.5 μg per rat twice a week, subcutaneouslyinjected) vs. vehicle-treated ovariectomized controls and vehicle-treated sham-operated controls. Uteri were removed,weighed and analysed under both the structural and histomorphometrical points of view.Key findings: Our data show that ferutinin acts, similarly to estradiol benzoate, on the uterus stimulating endometrialand myometrial hypertrophy; this notwithstanding, the phytoestrogen ferutinin, in contrast to estrogentreatment, appears to increase apoptosis in uterine luminal and glandular epithelia.Significance: Ferutinin, used in osteoporosis treatment primarily for bonemass recovering, seems in linewith aneventual protective function against uterine carcinoma, unlike estrogens so far employed in hormone replacementtherapy (HRT).


2011 - Effect of pulsed electromagnetic fields (PEMFs) on condrogenic phenotype maintenance of MSCs in presence of pro-inflammatory cytochines: preliminary results [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Benincasa, Marta; Sena, Paola; Gian Luigi, Sacchetti; Stefania, Setti; Cadossi, Ruggero
abstract

The aim of the present study was to evaluate in vitro the effects of PEMFs on maintenance over time of chondrocyte phenotype of conditioned Mesenchimal Stem Cells (MSCs), in presence of pro-inflammatory cytokines (IL-ß1). MSCs, taken from bone marrow, were pellet-cultured in medium conditioning towards the chondrogenic lineage. Two targets were pursued: the first was to standardize the method to obtain chondrocyte pellets in terms of type/amount of withdrawal, time/degree of differentiation and amount of extracellular matrix production; the second was to extend over time chondrocyte differentiation, checking the phenotype maintenance, after adding pro-inflammatory IL-ß1 cytokine in culture medium with/without the application of PEMFs (device provided by IGEA-Carpi). The pellets obtained were coltured for different times (21, 28, 34 days), verifying the presence of type-II collagen (as index of chondrocyte differentiation) both by means of TEM analysis and immunoreaction. The best differentiation was obtained after 28 days of culture; in such pellets the studies were performed in triplicate for 15 days, identifying four experimental conditions: 1) without IL-b1 and PEMFs; 2) with IL-b1, without PEMFs; 3) without IL-b1 and with PEMFs; 4) with IL-b1 and PEMFs. The parameters of applied PEMFs were 1.7mT and 75Hz, and the time of application was 4 hours/day. Medium was changed every 3-4 days and stored for the evaluations of PGE2 (indicative of inflammation) and proteoglycans (indicative of chondrogenic differentiation). At the end of the experiment, each pellet was fixed with paraformaldehyde 4% and embedded in paraffin; sections (5 µm thick) were obtained and stained with Toluidin Blue in order to evaluate metachromasia. The results indicate that: 1) only the pellets treated with IL-b1 without PEMFs did not show metachromasia, indicanting a chondrocyte de-differentiation towards fibroblastic phenotype; 2) only in pellets treated with IL-b1 and with PEMF application, after about 12 days of treatment the amount of PGE2 in medium decreases (31%) while the proteoglycan production slightly increases (2%).In conclusion, if the results will be confirmed, pulsed electromagnetic fields could be proposed in preventing chondrocyte de-differentiation due to inflammation induced by IL-ß1; this with the final aim to integrate regenerative medicine techniques to apply in the healing of joint cartilage lesions with bio-physic energy devices, in order to obtain a stable-in-time recovery of physiologic function of articular surfaces that suffered a severe injury.


2011 - INTERACTION OF BIOPHYSIC STIMULI ON CONDROGENIC DIFFERENTIATION OF MSCs: PRELIMINARY RESULTS ON THE EVALUATION OF ANTI-INFLAMMATORY EFFECT OF ELECTROMAGNETIC FIELDS [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Benincasa, Marta; Taronna, ANGELO PIO; Sena, Paola; Setti, S.; Cadossi, Ruggero
abstract

The aim of the present study is to investigate in vitro chondrocyte-like cells treated with electromagnetic fields to evaluate over time maintenance of chondrocyte phenotype, in presence of pro-inflammatory cytokines (IL-1ß). Mesenchimal Stem Cells taken from bone marrow were cultured (in pellet) in medium conditioning towards the chondrogenic lineage. The targets are firstly to standardize the method to obtain chondrocyte pellets in terms of a) type/amount of withdrawal, b) time/degree of differentiation, and c) amount of extracellular matrix production; secondly, to extend over time chondrocyte differentiation, checking the phenotype maintenance, after adding pro-inflammatory cytokines in culture medium with/without the application of electromagnetic fields (device provided by IGEA-Carpi). The pellets obtained were coltured for different times (21, 28, 34 days), verifying the presence of type 2 collagen (index of chondrocyte differentiation). The best differentiation was obtained after 28 days of culture. In such pellets, after inflammatory induction and application of electromagnetic field (1.7mT, 75Hz) for 15 days, the observations showed that after about 12 days of treatment the amount of PGE2 in medium decreases (31%) while the proteoglycan production slightly increases (2%). In conclusion, electromagnetic fields could be proposed (if the results will be confirmed) in preventing chondrocyte de-differentiation due to inflammation induced by IL-1ß, to integrate regenerative medicine techniques in the healing of cartilage lesions.


2011 - RGB method in immunofluorescence investigations on stem cells [Articolo su rivista]
Riccio, Massimo; E., Resca; Bertoni, Laura; Cavani, Francesco; Sena, Paola; Ferretti, Marzia; Baldini, Andrea; Palumbo, Carla; DE POL, Anto
abstract

Colour is not related to a particular discipline, but it is transversely present in many circles and inalmost all the aspects of life. It has a special value in art, but also as far as other disciplines areconcerned, like the sciences, the colour is at the basis of some of their intrinsic significances and it oftenneeded to allow the interpretation of some of their phenomena as well. As regards the development ofcell biology knowledge, colour acquired more and more importance in revealing the observations of theresearchers. A field in which the methods based on the colours are particularly employed is theimmunofluorescence, used to identify specific proteins in cells and tissues. These techniques combinethe fluorochrome properties with specific molecules, i.e. antibodies, directed against particularsubstances to investigate, for example a specific protein. In single immunofluorescence analysis, thesignal from an excited fluorochrome corresponds to a particular protein. In multiple immunofluorescenceanalysis, two or more signals are simultaneously detected to show the localization of differentproteins on the same sample. The three primary colours red, green and blue were currently assigned tothe signals from immunofluorescence-processed samples and visualized by the RGB method. In thepresent work, different examples of RGB applications in immunocytochemical investigations areshowed: the first concerns the multiple analysis of three markers, localized in different loci of the cellplasma membrane; the second is related to the co-localization of two signals in the same site of specificsubcellular structures. In this case the secondary colours, obtained by overlapping the primary ones,demonstrate the specific co-presence of two proteins in the same site. With the present paper, theauthors wish to underline the relevant role of colours also in those areas in which colours are the meansnot the end.


2010 - Decreased osteocyte viability in multiple myeloma patients: osteolytic bone lesions, apoptosis and their potential role in bone remodeling alterations. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Benincasa, Marta; Lazzaretti, Mirca; M., Abeltino; M., Bolzoni; C., Mancini; E., Martella; P., Storti; N., Giuliani
abstract

Osteocytes seem to regulate bone remodelling by different manners including apoptosis. A reduction of osteocyte viability (OC-V) was shown in osteoporotic bone. Osteolysis/osteoporosis, induced by multiple myeloma (MM), are characterized by severely imbalanced uncoupled bone remodelling due to increased osteoclastogenesis and suppressed osteoblast differentiation occurring close to MM cell infiltration. The aim of this study is to investigate the eventual involvement of osteocytes in bone remodelling alterations occurring in MM patients. Iliac crest biopsies were taken from 34 patients with MM (52% of which displayed osteolytic bone lesions), 10 with monoclonal gammopathy of uncertain significance (MGUS) and 10 without haematological malignancies/osteoporosis/metabolic bone diseases. Viable osteocytes and degenerated or apoptotic osteocytes/empty lacunae were evaluated on 500 lacunae per histological section. Significant reductions of OC-V in MM patients were found compared to healthy controls, whereas not statistical significance in OC-V was observed between MM and MGUS. Death osteocytes/empty lacunae number was significantly increased in MM vs. controls but not as compared to MGUS. Concerning the skeletal involvement, in MM patients either OC-V percentage was significantly lower in osteolytic vs. non-osteolytic patients or the number of dead osteocytes/empty lacunae was higher in osteolytic vs. non-osteolytic patients. Monolayers were also performed of human preosteocytes incubated with/without conditioned media (CM) taken from human myeloma cell lines (HMCLs) or co-cultured with them, and TEM observations showed dead cells in those monolayers treated with HMCL-CM or co-cultured with HMCLs as compared to non treated cells. In CM of preosteocytes co-cultured with HMCLs significantly increased CD14+-derived osteoclastogenesis occurs, evaluated by TRAP staining and pit-forming assay. Our data demonstrate that MM bone is characterized by reduction of OC-V; the increase of osteocyte death (apoptosis/degeneration) in relation to the presence of bone lesions may represent a triggering event to osteoclast recruitment.


2010 - Influence of ferutinin on bone metabolism in ovariectomized rats. II: Role in recovering osteoporosis. [Articolo su rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Zavatti, Manuela; Resca, Elisa; Carnevale, Gianluca; Benelli, Augusta; Zanoli, Paola; Palumbo, Carla
abstract

The study investigates the influence of ferutinin (a phytoestrogen extracted from Ferula Hermanis root) on bone metabolism in ovariectomized rats. The study represent the complection of previous investigations (published in 2009, concerning the ferutinine role in preventing osteoporosis due to estrogen deficiency). The present investigation concerns the role of Ferutinine in recovering osteoporosis.


2010 - New aspects of Ferutinin effect in preventing osteoporosis [Abstract in Rivista]
Ferretti, Marzia; Cavani, Francesco; Bertoni, Laura; Zavatti, Manuela; Taronna, ANGELO PIO; Carnevale, Gianluca; Benelli, Augusta; Zanoli, Paola; Marotti, Gastone; Palumbo, Carla
abstract

The results of the study suggest that ferutinin role, in preventing osteoporosis due to estrogen deficiency, is expressed in inhibiting osteoclast erosion rather than in enhancing osteoblast deposition (as previously suggested); moreover, in all F-OVX groups the bone turnover is very low and seems correlated to the trivial body weight increase, which, in turn, depends on ferutinin treatment.


2010 - STATIC OSTEOGENESIS AND DYNAMIC OSTEOGENESIS: THEIR RELEVANCE IN DENTAL BONE IMPLANTS AND BIOMATERIAL OSSEOINTEGRATION [Articolo su rivista]
Marotti, Gastone; Zaffe, Davide; Ferretti, Marzia; Palumbo, Carla
abstract

The present report summarizes the results of a series of investigations carried out in our laboratory on intramembranous ossification occurring under normal condition during skeletal organogenesis and osseointegrations of dental implants and biomaterials. No morphological differences were observed between normal and pathological conditions, since the same following sequence of events were found. Inside the embryonic mesenchyme or the connective tissue formed after bleeding, due to surgery, cords of plum cells, displaying the typical osteoblastic structure, differentiate in between the blood capillaries. These osteoblasts appear to be stationary since they do not move, but transform into osteocytes in the same site where they differentiated, thus giving origin to a trabecular woven-bone framework laid down by static osteogenesis (SO). Soon after, typical movable osteoblastic laminae differentiate along the surface of this SO-trabeculae and thicken them with lamellar bone formed by dynamic osteogenesis (DO). SO seems to depend on inductive stimuli and appears to be mechanically independent, whereas DO mainly depend on mechanical strains. Additionally SO-bone is a bad quality bone because of its woven texture and high microporosity, due to the many osteocyte lacunae it contains, whereas DO-bone generally is a lamellar bone and thus mechanically much more resistant.The clinical implication of these findings, as regards the time of load application after prostheses/biomaterials implantation, is discussed.


2009 - Bone tissue electroporation: a preliminary in vivo study. [Abstract in Rivista]
Cavani, Francesco; Fini, M.; Bertoni, Laura; Ferretti, Marzia
abstract

Goal of the study is to extablish the values of parameners of electroporation above which, in the treated bone regions, cell damage becomes irreversible, leading to cell death.


2009 - Effect of leptin on the development of primary ossification centers in mouse fetuses. [Abstract in Rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Zavatti, Manuela; Benelli, A.; Palumbo, Carla
abstract

Leptin may be considered a significat cartilage/bone growth factor.


2009 - Influence of ferutinin on bone metabolism in ovariectomized rats. I: role in preventing osteoporosis [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Resca, Elisa; Casolari, Barbara; Carnevale, Gianluca; Zavatti, Manuela; C., Montanari; Benelli, Augusta; Zanoli, Paola
abstract

Phytoestrogens play a role in maintaining bone mass in the post-menopausal period for their putative function as osteoprotective agents. The aim of the present study was to investigate the influence of Ferutinin, a phytoestrogen found in the plants of Ferula genus, on bone loss in ovariectomized rats. Such an animal model can simulate the various clinical syndromes deriving from osteoporosis. The effect of the daily oral administration of ferutinin to ovariectomized rats (dosed at 2 mg/kg per day for 30 and 60 days) was compared to that of estradiol benzoate (subcutaneously administered at the dose of 1.5 microg/rat twice a week). After the sacrifice, histomorphometrical analyses were performed on trabecular bone of L4-L5 vertebrae and distal femoral metaphysis, as well as on cortical bone of femoral diaphysis; biochemical parameters (bone mineral components and markers) were also evaluated from the rat serum. The histomorphometrical analyses of trabecular and cortical bone from lumbar vertebrae and femur showed that ferutinin has the same antiosteoporotic effect of estradiol benzoate on bone mass, and in some cases is even stronger. This fact suggests that it could prevent osteoporosis caused by severe estrogen deficiency in ovariectomized rats. The possibility of using ferutinin as an alternative to the commonly employed hormonal replacing therapy in post-menopausal women is discussed.


2009 - Leptin increases growth of primary ossification centers in fetal mice [Articolo su rivista]
Bertoni, Laura; Ferretti, Marzia; Cavani, Francesco; Zavatti, Manuela; Resca, Elisa; Benelli, Augusta; Palumbo, Carla
abstract

The effect of peripheral leptin on fetal primary ossification centers during the early phases of bone histogenesis was investigated by administration of leptin to pregnant mice. Fourteen pregnant mice were divided into two groups. The treated pregnant group was subcutaneously injected in the intrascapular region with supraphysiologic doses (2 mg kg(-1)) of leptin (Vinci Biochem, Firenze, Italy) in a volume of 0.1 mL per 10 g body weight, at the 7th, 9th and 11th day of gestation. The control group was treated with physiological solution in the same manner and same times as the treated group. The new-born mice were killed 1 day after birth and the primary ossification centers were stained with Alizarin Red S after diaphanizing the soft tissues in 1% potassium hydroxide. The development of both endochondral and intramembranous ossification centers was morphometrically analysed in long bones. The results showed that the ossification centers of mice born by mothers treated with leptin grow more rapidly in both length and cross-sectional area compared with mice born by the untreated mothers. As the development of long bones depends on endochondral ossification occurring at proximal and distal epiphyseal plates as well as on intramembranous ossification along the periosteal surface, it appears that leptin activates the differentiation and proliferation of both chondrocytes and osteoblasts. The role of leptin as a growth factor of cartilage and bone is discussed in the light of the data reported in the literature.


2008 - Bisphosphonate-associated jawbone osteonecrosis: a correlation between imaging techniques and histopathology. [Articolo su rivista]
Alberto, Bedogni; Stella, Blandamura; Zerina, Lokmic; Palumbo, Carla; Mirko, Ragazzo; Francesca, Ferrari; Alberto, Tregnaghi; Francesco, Pietrogrande; Olindo, Procopio; Giorgia, Saia; Ferretti, Marzia; Giorgio, Bedogni; Chiarini, Luigi; Giuseppe, Ferronato; Vito, Ninfo; Lucio Lo, Russo; Lorenzo Lo, Muzio; Pier Francesco, Nocini
abstract

Objectives. Recently, jawbone osteonecrosis has been reported as a potential adverse effect of bisphosphonates administration. This paper considers and highlights histopathologic and radiologic features of this condition. Study design. Eleven patients, owing to unresponsiveness to conservative treatment and uncontrollable pain, underwent surgical resection of diseased jawbone after extensive hyperbaric oxygen therapy. A thorough clinical, laboratory, and imaging study was performed. Surgical specimens underwent histopathologic and immunohistochemical evaluation. Results. Computerized tomography (CT) scans showed increased bone density, periosteal reaction, and bone sequestration in advanced stages. With magnetic resonance imaging (MRI), exposed areas showed a low signal in T1-and T2-weighted and inversion recovery images, which suggests low water content and is histopathologically correlated with paucity in cells and vessels (osteonecrotic pattern). Unexposed diseased bone was characterized by T1 hypointensity and T2 and IR hyperintensity, which suggests high water content and inflammation, associated with hypercellularity, osteogenesis, and hypervascularity (osteomyelitic pattern). Conclusions. Diseased bone extends beyond the limits of the bone exposed in the oral cavity. Histopathologic examination correlated well with CT and MRI, which are the choice for the evaluation of bisphosphonate-associated jawbone osteonecrosis.


2008 - Effect of the phytoestrogen ferutinin in preventing and recovering osteoporosis: histomorphometric analysis of bone mass in ovariectomized rats. [Abstract in Rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Resca, Elisa; Carnevale, Gianluca; Zavatti, Manuela; Benelli, A.; Zanoli, P.; Palumbo, Carla
abstract

Ferutinin seems to display the same effects on bone mass obtained with estradiol in OVX rats.


2008 - Human dental pulp stem cells (HDPSC) versus osteoblast-like cells: comparison for capability of adhesion, growth and bone matrix formation on differently-shaped surfaces of biomaterials. [Abstract in Rivista]
Palumbo, Carla; Riccio, M.; Resca, E.; Bretoni, L.; Ferretti, Marzia; Cavani, Francesco; Bruzzesi, G.; Baldini, Andrea; DE POL, Anto
abstract

Comparisons between HDPSC and osteoblast-like cells are made in regards to bone matrix production as well as distribution, density and adhesion to the biomaterial surfaces.


2008 - Influence of ferutinin on bone mass and its side effects in ovariectomized rats. [Abstract in Rivista]
Ferretti, Marzia; Palumbo, Carla; Bretoni, L.; Cavani, Francesco; Resca, E.; Benincasa, Marta; Carnevale, Gianluca; Zavatti, Manuela; Montanari, C.; Benelli, A.; Zanoli, P.; Marotti, Gastone
abstract

Ferutinin seems to display the same effects on bone mass recorded with estradiol, but with respect to estrogens it seems to extert a protection against uterine carcinoma.


2008 - Ovariectomy sensitizes rat cortical bone to whole-body vibration [Articolo su rivista]
A., Rubinacci; M., Marenzana; Cavani, Francesco; F., Colasante; I., Villa; J., Willnecker; G. L., Moro; L. P., Spreafico; Ferretti, Marzia; F., Guidobono; Marotti, Gastone
abstract

This study was designed to determine the modulatory effect of estrogen on mechanical stimulation in bone. Trabecular and cortical bone compartments of ovariectomized rats exposed to whole-body vibration of different amplitudes were evaluated by peripheral quantitative computed tomographic (pQCT) analysis and histomorphometry and compared to controls not exposed to vibration. Rats underwent whole-body vibration (20 minutes/day, 5 days/week) on a vibration platform for 2 months. The control rats were placed on the platform without vibration for the same time. We divided rats into six groups: a sham control (SHAM); a sham vibrated (SHAM-V) at 30 Hz, 0.6 g; a SHAM-V at 30 Hz, 3g; an ovariectomized control (OVX); an ovariectomized vibrated (OVX-V) at 30 Hz, 0.6 g; and an OVX-V at 30 Hz, 3g. In vivo, pQCT analyses of the tibiae were performed at the start of the experiment and after 4 and 8 weeks. After 8 weeks the tibiae were excised for histomorphometric and for in vitro pQCT analyses. In the SHAM-V group, vibration had no effect upon the different bone parameters. In the OVX-V group, vibration induced a significant increase compared to the OVX group of the cortical and medullary areas (P < 0.01) and of the periosteal (P < 0.01) and endosteal (P < 0.05) perimeters at the 3 g vibration. The strain strength index increased in the OVX-V group significantly (P < 0.01) at the higher vibration. The results showed that low-amplitude, high-frequency whole-body vibration is anabolic to bone in OVX animals. The osteogenic potential is limited to the modeling of the bone cortex and depends on the amplitude of the vibration.


2008 - Sympathectomy alters bone architecture in adult growing rats [Articolo su rivista]
F., Pagani; V., Sibilia; Cavani, Francesco; Ferretti, Marzia; Bertoni, Laura; Palumbo, Carla; N., Lattuada; E., De Luca; A., Rubinacci; F., Guidobono
abstract

Sympathetic nervous system (SNS) fibres and alpha- and beta-receptors are present in bone, indicating that the SNS may participate in bone metabolism. The importance of these observations is controversial because stimulation or inhibition of the SNS has had various effects upon both anabolic and catabolic activity in this tissue. In this study we evaluated the effects of pharmacological sympathectomy, using chronic treatment of maturing male rats with 40 mg of guanethidine/kg i.p., upon various parameters in bone. Double labelling with tetracycline injection was also performed 20 and 2 days before sacrifice. Bone mass, mineral content, density and histomorphometric characteristics in different skeletal regions were determined. Bone metabolic markers included urinary deoxypyridinoline and serum osteocalcin measurements. Guanethidine significantly reduced the accretion of lumbar vertebral bone and of mineral content and density, compared to controls. Femoral bone mineral content and density were also significantly reduced, compared to controls. Histomorphometric analyses indicated these effects were related to a reduction of cortical bone and mineral apposition rate at femoral diaphysials level. Both markers of bone metabolism were reduced in controls as they approached maturity. Guanethidine significantly decreased serum osteocalcin compared to controls, while urinary deoxypyridinoline was unchanged. These data indicate that guanethidine-induced sympathectomy caused a negative balance of bone metabolism, leading to decreased mass by regulating deposition rather than resorption during modeling and remodeling of bone.


2008 - Two peculiar conditions following a coma: A clinical case of heterotopic ossification concomitant with keloid formation [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Pierluigi, Bonucci; Sena, Paola; Bertoni, Laura; Cavani, Francesco; Andrea, Celli; Rovesta, Claudio
abstract

The etiology and formation pattern of heterotopic ossifications (HO) are still unknown. They occur in soft tissues in which bone does not normally form, near one or more proximal joints. In this article, the authors report a peculiar case of a 31-year-old patient affected by scapulo-humeral ankylosis that occurred about 6 months after a coma, in which two unusual concomitant conditions were observed: HO formation in the scapulo-humeral region and the development of keloids during wound repair. The scapulo-humeral ankylosis was resolved surgically with the removal of the HO, which was then studied morphologically to understand its formation pattern. By light microscopy and transmission electron microscopy, it was observed that heterotopic bone displays the normal microscopic structure of primary bone, in which two types of bone tissue were recognized, i.e., woven-fibered bone, deeply located and produced first, and lamellar bone. This suggests that the pattern of HO formation retraces the ontogenetic steps that normally occur during intramembranous ossification. The authors also discuss the peculiar concomitance of HO formation and keloid development, speculating that, although they are different conditions localized in dissimilar regions, they might be hypothetically triggered by a common event, such as the release of factors likely issued during the coma status.


2007 - Phytoestrogen effects on bone mass in ovariectomized rats: preliminary histomorphometric analysis. [Abstract in Rivista]
Ferretti, Marzia; Palumbo, Carla; Cavani, Francesco; Bertoni, Laura; Resca, E.; Carnevale, Gianluca; Zavatti, Manuela; Montanari, C.; Benelli, A.; Zanoli, P.; Marotti, Gastone
abstract

Phytoestrogens ferutinine could prevent in rats the risk of osteoporosis in estrogen deficient conditions and it could enhance the recover of bone mass in osteoporotic OVX rats.


2006 - Different skeletal regional response to continuous brain infusion of leptin in the rat. [Articolo su rivista]
F., Guidobono; F., Pagani; V., Sibila; C., Netti; N., Lattuada; D., Rapetti; E., Mrak; I., Villa; Cavani, Francesco; Bertoni, Laura; Palumbo, Carla; Ferretti, Marzia; Marotti, Gastone; A., Rubinacci
abstract

This study was designed to evaluate whether or not continuous intracerebroventricular infusion of leptin (1.5 mu g/rat/24 h, for 28 days) produced different regional response on the skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content (BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydincline and serum osteocalcin remained more elevated in the leptin-treated group as compared to controls. The results demonstrate that long-term central infusion of leptin activates bone remodeling with a negative balance. Leptin induces distinct responses in the different structure of bone and in the axial and appendicular skeleton. (c) 2005 Elsevier Inc. All rights reserved.


2006 - Does Static precede dynamic osteogenesis in endochondral ossification as occurs in intramembranous ossification? [Articolo su rivista]
Ferretti, Marzia; Palumbo, Carla; Bertoni, Laura; Cavani, Francesco; Marotti, Gastone
abstract

Endochondral ossification takes place with calcified cartilage cores providing a rigid scaffold for new bone formation. Intramembranous ossification begins in connective tissue and new bone formed by a process of static ossification (SO) followed by dynamic ossification (DO) as previously described. The aim of the present study was to determine if the process of endochondral ossification is similar to that of intramembranous ossification with both a static and a dynamic phase of osteogenesis. Endochondral ossification centers of the tibiae and humeri of newborn and young growing rabbits were studied by light and transmission electron microscopy. The observations clearly showed that in endochondral ossification, the calcified trabeculae appeared to be lined first by osteoclasts. The osteoclasts were then replaced by flattened cells (likely cells of the reversal phase) and finally by irregularly arranged osteoblastic laminae, typical of DO. This cellular sequence did not include osteoblasts seen in the phase of SO. These findings clearly support our working hypothesis that SO only forms in soft tissues to provide a rigid framework for DO, and that DO requires a rigid mineralized surface. The presence of osteocytes in contact with the calcified cartilage also suggests the existence of stationary osteoblasts in endochondral ossification. Stationary osteoblasts did not appear to be a unique feature of SO. The presence of stationary osteoblasts may appear to provide the initial osteocytes during osteogenesis that may function as mechanosensors throughout the bone tissue. If this is the case, then bone would be capable of sensing mechanical strains from its inception.


2006 - Leptin effect on rat primary ossification centers during bone histogenesis. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Benincasa, Marta; Bertoni, Laura; Cavani, Francesco; Rivasi, M.; Benelli, A.
abstract

During the early phases of endochondral ossification, Leptin positive effects are shown in growith of rat ossification centers.


2006 - Preliminary observations on transplants of vascularized bone scaffolds: an experimental and morphological study. [Abstract in Rivista]
Ferretti, Marzia; Palumbo, Carla; Bertoni, Laura; Cavani, Francesco; Carbonara, A.; DE SANTIS, Giorgio; Marotti, Gastone
abstract

Bone formation occurring inside and around dead bone implanted scaffolds seems to follow the same sequence of events that are observed during normal intramembranous ossification.


2005 - Endochordral versus intramembranous ossification: analogies and differences. [Abstract in Rivista]
Ferretti, Marzia; Palumbo, Carla; Bertoni, Laura; Marotti, Gastone
abstract

Stationary osteoblasts are not a typical finding of static osteogenesis; they may also exist in the earòly stage of dynamic osteogenesis.


2005 - Heterotopic human bone: mechanism of deposition and structure. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Rovesta, Claudio
abstract

Heterotopic human bone is a pathologic bone that needs to degenerate rather than to be preserved, and for this reason rapidly osteocyte death occurs.


2004 - Endotendinous ossification and static osteogenesis: analogies and differences. [Abstract in Rivista]
Palazzini, S.; Palumbo, Carla; Ferretti, Marzia; Zaffe, Davide; Marotti, Gastone
abstract

In endotendinous ossification and in the early phase (SO) of intramembranous ossification the first trabecular framework fiorms in a different manner.


2004 - In vivo leptin expression in cartilage and bone cells of growing rats and adult humans [Articolo su rivista]
M., Morroni; R., De Matteis; Palumbo, Carla; Ferretti, Marzia; I., Villa; A., Rubinacci; S., Cinti; Marotti, Gastone
abstract

The present investigation was carried out to analyse, immunohistochemically, in vivo leptin expression in cartilage and bone cells, the latter restricted to the elements of the osteogenic system (stromal cells, osteoblasts, osteocytes, bone lining cells). Observations were performed on the first lumbar vertebra, tibia and femur of four rats and on the humerus, femur and acromion of four patients. Histological sections of paraffin-embedded bone samples were immunostained using antibody to leptin. The results showed that, in growing rat bone, leptin is expressed in chondrocytes and stromal cells, but not in osteoblasts; bone lining cells were not found in the microscopic fields examined. In adult human bone, leptin is expressed in chondrocytes, stromal cells and bone lining cells; osteoblasts were not found in the microscopic fields examined. Osteocytes were found to be leptin positive only occasionally and focally in both rat and human bone. The in vivo findings reported show, for the first time, that leptin appears to be expressed only in the cells of the osteogenic lineage (stromal cells, bone lining cells, osteocytes) that, with respect to osteoblasts, are permanent and inactive, i.e. in those cells that according to our terminology constitute the bone basic cellular system (BBCS). Because the BBCS seems to be primarily involved in sensing and integrating mechanical strains and biochemical factors and then in triggering and driving bone formation and/or bone resorption, it appears that leptin seems to be mainly involved in modulating the initial phases of bone modelling and remodelling processes.


2004 - Leptin expression in the cells of the osteogenic lineage of growing rat and adult human bones. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; De Matteis, R.; Morroni, M.; Marotti, Gastone
abstract

Leptin seems to be involved in modulating the initial phases of bone modeling and remodeling processes.


2004 - Osteocyte dendrogenesis in static and dynamic bone formation: An ultrastructural study [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Marotti, Gastone
abstract

The present ultrastructural investigation into osteocyte dendrogenesis represents a continuation of a previous study (Ferretti et al., Anat. Embryol., 2002; 206:21-29), in which we pointed out that, during intramembranous ossification, the well-known dynamic bone formation (DBF), performed by migrating osteoblast laminae, is preceded by static bone formation (SBF), in which cords of stationary osteoblasts transform into osteocytes in the same site where they differentiated. The research was carried out on the perichondral center of ossification surrounding the mid shaft level of various long bones of chick embryos and newborn rabbits. Transmission electron microscope observations showed that the formation of osteocyte dendrites is quite different in the two types of osteogenesis, mainly depending on whether or not osteoblast movement occurs. In DBF, osteoblasts transform into small ovoidal/ellipsoidal osteocytes and their dentrites form in an asynchronous and asymmetrical manner in concomitance with, and depending on, the advancing mineralizing surface and the receding osteogenic laminae. In SBF, stationary osteoblasts give rise to big globous osteocytes, located inside confluent lacunae, with short and symmetrical dendrites that can radiate simultaneously all around their cell body because they are completely surrounded by unmineralized matrix. Contacts and gap junctions were observed between all osteocytes (both SBF- and DBF-derived) and between osteocytes and osteoblast's. Finally, a continuous osteocyte network extends throughout the bone, regardless of its static or dynamic origin. This network has the characteristic of a functional syncytium, potentially capable of modulating, by wiring transmission, the cells of the osteogenic lineage covering the bone surfaces. (C) 2004 Wiley-Liss, Inc.


2004 - Static and dynamic osteogenesis in bone regeneration and repair. [Abstract in Rivista]
Marotti, Gastone; Palumbo, Carla; Ferretti, Marzia; Cavani, Francesco; Botti, P.; Cadossi, Ruggero; Cane, V.
abstract

Static osteogenesis probably depends on inductive factors, whereas dynamic osteogenesis more likely is mailly conditioned by mechanical signals.


2003 - Apoptosis during intramernbranous ossification [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; DE POL, Anto
abstract

This paper concerns the role of apoptosis during the onset of bone histogenesis. Previous investigations by us performed on intramembranous ossification revealed the existence of two types of osteogenesis: static (SBF) and dynamic bone formation (DBF). During SBF, the first to occur, stationary osteoblasts transform into osteocytes in the same location where they differentiated, forming the primary spongiosa. DBF takes place later, when movable osteoblastic laminae differentiate along the surface of the primary trabeculae. The main distinctive feature between SBF and DBF is that the latter involves the invasion of pre-existing adjacent tissue, whereas the former does not. To ascertain whether programmed cell death during the invasive DBF process determines the fate of surrounding pre-existing mesenchyme differently from that occurring during the non-invasive SBF process, we studied apoptosis in ossification centres of tibial diaphysis in chick embryos and newborn rabbits with TUNEL and TEM. It emerged that, in both SBF and DBF, apoptosis affects mesenchymal cells located between the forming trabeculae and capillaries. However, apoptotic cells were observed more frequently during DBF than during SBF. This suggests that, during bone histogenesis, apoptosis, which is mostly associated with the invasive process of DBF, is probably dedicated to making space for advancing bone growth.


2003 - Apoptosis of striate muscle fibres after tendon lesions: preliminary observations. [Abstract in Rivista]
Palumbo, Carla; Rovesta, Claudio; Leigheb, M.; Ferretti, Marzia; DE POL, Anto
abstract

Tendon lesions imply the degeneration of the injuried tendon that, in its turn, induces the partial disuse of the muscle mass that undergoes atrophy by apoptosis.


2002 - Apoptosis during static and dynamic bone formation. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; DE POL, Anto; Marotti, Gastone
abstract

The study shows that apoptotic phenomena occur in cells of fibroblastic type, located between the newly-forming bony trabeculae and blood capillaries; however, the number of apoptotic cells is incomparably higher during dynamic bone formation than static bone formation.


2002 - Avian scleral bones and human auditory ossicles, two different models to study osteocyte apoptosis in relation with bone remodeling. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; DE POL, Anto
abstract

Osteocyte death could represent a programmed phenomenon (apoptosis) in those skeletal segments that should not undergo bone remodeling, because are submitted to steriotyped mechanical stresses for the whole life.


2002 - Bone as an ionic exchange system: evidence for a link between mechanotransduction and metabolic needs [Articolo su rivista]
A., Rubinacci; M., Covini; C., Bisogni; I., Villa; M., Galli; Palumbo, Carla; Ferretti, Marzia; Muglia, Maria Antonietta; Marotti, Gastone
abstract

To detect whether the mutual interaction occurring between the osteocytes-bone lining cells system (OBLCS) and the bone extracellular fluid (BECF) is affected by load through a modification of the BECF-extracellular fluid (ECF; systemic extracellular fluid) gradient, mice metatarsal bones immersed in ECF were subjected ex vivo to a 2-min cyclic axial load of different amplitudes and frequencies. The electric (ionic) currents at the bone surface were measured by a vibrating probe after having exposed BECF to ECF through a transcortical hole. The application of different loads and different frequencies increased the ionic current in a dose-dependent manner. The postload current density subsequently decayed following an exponential pattern. Postload increment's amplitude and decay were dependent on bone viability. Dummy and static loads did not induce current density modifications. Because BECF is perturbed by loading, it is conceivable that OBLCS tends to restore BECF preload conditions by controlling ion fluxes at the bone-plasma interface to fulfill metabolic needs. Because the electric current reflects the integrated activity of OBLCS, its evaluation in transgenic mice engineered to possess genetic lesions in channels or matrix constituents could be helpful in the characterization of the mechanical and metabolic functions of bone.


2002 - Osteocyte dendrogenesis in static bone formation. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; DE POL, Anto; Marotti, Gastone
abstract

All osteocytes, independently of their SBF or DBF origin, take part in the formation on the continuous osteocytic network, connected by electric synapsis (gap junctions), potentially capable of modulating by wiring transmission the cells covering the bone surfaces.


2002 - Static and dynamic osteogenesis: two different types of bone formation [Articolo su rivista]
Palumbo, Carla; Contri, Miranda; Marotti, Gastone; Ferretti, Marzia
abstract

The onset and development of intramembranous ossification centers in the cranial vault and around the shaft of long bones in five newborn rabbits and six chick embryos were studied by light (LM) and transmission electron microscopy (TEM). Two subsequent different types of bone formation were observed. We respectively named them static and dynamic osteogenesis, because the former is characterized by pluristratified cords of unexpectedly stationary osteoblasts, which differentiate at a fairly constant distance (28+/-0.4 mum) from the blood capillaries, and the latter by the well-known typical monostratified laminae of movable osteoblasts. No significant structural and ultrastructural differences were found between stationary and movable osteoblasts, all being polarized secretory cells joined by gap junctions. However, unlike in typical movable osteoblastic laminae, stationary osteoblasts inside the cords are irregularly arranged, variously polarized and transform into osteocytes, clustered within confluent lacunae, in the same place where they differentiate. Static osteogenesis is devoted to the building of the first trabecular bony frame-work having, with respect to the subsequent bone apposition by typical movable osteoblasts, the same supporting function as calcified trabeculae in endochondral ossification. In conclusion, it appears that while static osteogenesis increases the bone external size, dynamic osteogenesis is mainly involved in bone compaction, i.e., in filling primary haversian spaces with primary osteons.


2002 - The osteocyte-bone lining cell system as transducer of mechanical strains into biological signals. [Abstract in Rivista]
Rubinacci, A; Covini, M; Bisogni, C; Villa, I; Palumbo, Carla; Ferretti, Marzia; Marotti, Gastone
abstract

The study shows that OBLCS tends to restore BECF preload conditions by controlling ion fluxes at the bone-plasma interface, thus playing a pivotal role in mechano-transduction and mineral homeostasis.


2002 - TUNEL REVEALS THE ONSET OF APOPTOSIS ON MESENCHYMAL CELLS IN STATIC AND DYNAMIC BONE FORMATION. [Abstract in Rivista]
Ferretti, Marzia; Palumbo, Carla; Benincasa, Marta; DE POL, Anto
abstract

Apoptosis is mostly associated with DBF with respect SBF during intramembranous ossification.


2001 - Apoptosis during intramembranous ossification: ultrastructural observations. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Marzona, Laura; DE POL, Anto
abstract

During intramembranous ossification cell apoptosis occurs in the periosteal fibrous tissue in parallel to bone growth and in association with blood vessels.


2001 - Osteocyte-bone lining cell system responds to cyclic loading in a dose dependent manner [Abstract in Rivista]
Rubinacci, A.; Covini, M.; Bisogni, C.; Villa, I.; Galli, M.; Palumbo, Carla; Ferretti, Marzia; Ardizzoni, Andrea; Marotti, Gastone
abstract

The mutual interaction between osteocyte-bone lining cell system (OBLCS) and the surrounding bone extracellular fluid which feels the continuous network of lacuno-canalicular microcavities plays a role in mechanotransduction. The study shows that OBLCS responds to cyclic loading depending on the applied loading parameters in a dose- dependent manner.


2001 - Osteocyte-osteoclast morphological relationships and the putative role of osteocyte in bone remodeling [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; A., Ardizzoni; Zaffe, Davide; Marotti, Gastone
abstract

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.


2001 - Osteocyte-osteoclast morphological relationships and the putative role of osteocytes in bone remodeling. [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Ardizzoni, Andrea; Zaffe, Davide; Marotti, Gastone
abstract

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60\% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.


2000 - Do osteocytes intervene in regulating osteoclast activity?. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Zaffe, Davide; Marotti, Gastone
abstract

Osteocyte could intervene in the process of bone resorption.


1999 - Histomorphometric study on the osteocyte lacuno-canalicular network in animals of different species. II. Parallel-fibered and lamellar bones. [Articolo su rivista]
Ferretti, Marzia; Muglia, M. A.; Remaggi, F.; Cane, V.; Palumbo, Carla
abstract

Osteocyte shape, size and density seem to depend mainly on collagen fiber texture rather than on the animal species.


1999 - Osteocyte-osteoclast morphological and putative functional relationships [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Zaffe, Davide; Marotti, Gastone
abstract

Osteocytes could intervene in the process of bone resorption.


1999 - Static and dynamic bone formation and the mechanism of collagen fiber orientation. [Abstract in Rivista]
Marotti, Gastone; Ferretti, Marzia; Palumbo, Carla; Benincasa, Marta
abstract

Woven bone forms by FBS, whereas the deposition of a bone tissue with orderly arranged collagen fibers (parallel-fibered and lamellar bone) may only occur by DBF, i.e. by movable osteoblasts.


1998 - Histomorphometric study on the osteocyte lacuno-canalicular network in animals of different species. I. Woven-fibered and parallel-fibered bones. [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Remaggi, Francesca; Canè, Valerio
abstract

A comparative histomorphometric study was carried out on the extension of lacuna-canalicular network in two types of bone tissue, i.e. woven-fibered and parallel-fibered, in shaft bones of various animals (frog, chicken, rabbit, dog, bovine, horse and man), with the aim to understand whether the distribution of osteocyte network is mainly related to the organization of the collagen fibres or to the animal species.


1998 - Osteocyte-bone lining cell system at the origin of steady ionic current in damaged anphibian bone. [Articolo su rivista]
A., Rubinacci; I., Villa; F. D., Benelli; E., Borgo; Ferretti, Marzia; Palumbo, Carla; Marotti, Gastone
abstract

A wound-generated steady electric current was measured by a two-dimensional vibrating probe system in the metatarsal banes of 22 adult frogs (Xenopus laevis) placed in amphibian Ringer. Inward currents were recorded entering a micrometric hole drilled through the cortex at middiaphyseal level. These steady state currents (mean +/- SD 8.50 +/- 2.77 mu A/cm(2)) last approximately 2 hours, were dependent on the presence of sodium in the incubation medium, were no more detectable after fixation, and were reduced to background level when the cell membranes were solubilized. These results agree with previous recordings of metatarsal bones of weanling mice, under identical conditions. Both results suggest that the measured ionic currents have a cellular origin. Metatarsal bones of adult amphibian were purposely selected for this study because, unlike mammalian bones, their shafts are avascular and only contain an osteocyte-bone lining fell system, as documented by scanning and transmission electron observations. Thus, unlike the data from previous investigations on mammals, the results succeeded in giving the first convincing evidence that the osteocyte-bone lining cell system is the origin of damage-generated ionic currents. As damage exposes bone ionic compartment to plasma, damage-generated ionic currents are representative of ion fluxes at bone plasma interface, and cells at the origin of the current generate the driving force of such fluxes. By demonstrating that osteocytes and bone lining cells are at the origin of the current, this study suggests that the osteocyte-bone lining cell system, though operating as a cellular membrane partition, regulates ionic flow between bone and plasma. Since strain-related adaptive remodeling could also depend on ionic characteristics and flow of the bone fluid through the osteocyte lacuno-canalicular network, the results reported here support the view that osteocyte and bone lining cells may constitute a functional syncytium involved in mineral homeostasis as well as in bone adaptation to mechanical loading.


1998 - Stromal cell structure and relationships in perimedullary spaces of chick embryo shaft bones [Articolo su rivista]
S., Palazzini; Palumbo, Carla; Ferretti, Marzia; Marotti, Gastone
abstract

Structure and relationships of stromal cells were studied by light (LM) and transmission electron microscopy (TEM) in the perimedullary spaces that form the growing cortex of the chick embryo tibia. Observation under LM showed that in all perimedullary spaces the interstices between the cells carpeting the bone surface and the endothelial Lining contain stromal cells surrounded by an amorphous matrix. Two types of stromal cells were distinguished: stellate and spindle-shaped. All stromal cells are alkaline phosphatase-positive. TEM showed that both types of stromal cells have cytoplasmic processes of Various length and calibre, coming into contact with each other as well as with endothelial. cells and osteoblasts or bone lining cells. Capillaries were found to have a continuous endothelial lining; occasionally endothelial cells radiate cytoplasmic processes towards stromal cells. Along all the above-mentioned cellular contacts adherens and/or gap junctions were often observed. The results of the present study, together with our previous findings on osteoblast-osteocyte relationships, show that the cells of the osteogenic lineage form a continuous protoplasmic network that extends from the osteocytes to the vascular endothelium, passing through osteoblasts (or bone lining cells) and stromal cells. The occurrence of gap junctions among this cytoplasmic network, namely of junctions enabling metabolic and electric coupling, indicates that it forms a functional syncytium, suggesting the hypothesis that the activity of the cells pertaining to the osteogenic Lineage might be regulated not only by diffusion (volume transmission) through the intercellular fluids of systemic (hormones) and local factors (cytokines, etc.) but also by signals issued through the cytoplasmic network of the osteogenic cells (wiring transmission).


1996 - Histomorphological and chemico-physical analyses of the mineral matrix of micropetrotic human bone [Articolo su rivista]
Remaggi, Francesca; Ferretti, Marzia; Canè, Valerio; Zaffe, Davide
abstract

Micropetrotic areas of human bone were analyzed with reference to their distance from blood vessels and to the age of the subjects. Undecalcified bone sections were treated with various solvent, soaking and etching substances to establish the nature of the material occluding the osteocytic canalicular cavities, and were examined by means of microradiographic and microdurimetric methods and X-ray microanalysis to evaluate the degree of mineralization in the bone matrix. Since it was only after strong etching with HCl that the canalicular network became visible under light and scanning electron microscopy, it is clear that the debris filling the-canalicular network consists almost entirely of mineral substance. The degree of mineralization of micropetrotic bone is high because it is always a more mature type Of bone, but the mineral content of the matrix and the Ca/P ratio do not differ significantly from those of neighbouring bone where the canalicular network is fully pervious.


1996 - Intermittent compressive load stimulates osteogenesis and improves osteocyte viability in bone coltured “in vitro”. [Articolo su rivista]
E., Lozupone; Palumbo, Carla; A., Favia; Ferretti, Marzia; Palazzini, Silvana; F. P., Cantatore
abstract

The paper clearly shows the effect of intermittent compressive load in stimulating osteogenesis and improving osteocyte viability in bone coltured “in vitro”.


1995 - Morphometric study of collagen maturation in chick compact bone [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Palazzini, Silvana; Zaffe, Davide
abstract

An ultrastructural-morphometric study was carried out on the process of osteoid maturation in growing surfaces of parallel-fibered chick bone. The aim was to investigate the distribution, size and amount of collagen fibrils (CFs), as well as the proteoglycan (PG) content, throughout the osteoid seam and in the adjacent bone. The results show that the organic components secreted by osteoblasts undergo complete maturation inside the osteoid seam only. Proceeding from the secreting plasma membrane of osteoblasts (osteoidogenic surface) towards the mineralizing surface, we found that CFs gradually increase in diameter but not in number per surface unit. As a consequence, the proportion of osteoid seam occupied by CF increases too, at the expense of the interfibrillar substance. PG content also decreases inversely in this direction. In the adjacent bone, CF size and density do not change significantly with respect to the mature osteoid close to the mineralizing surface.


1995 - Quantitative evaluation on osteocyte canalicular density in human secondary osteons. [Articolo su rivista]
Marotti, Gastone; Ferretti, Marzia; Remaggi, Francesca; Palumbo, Carla
abstract

Osteocyte canalicular density (OCD) was evaluated at different levels of the wall of human secondary osteons, in subjects of different ages, to find out whether any correlation exists between the extension of the canalicular network and the exponential decrement of the appositional growth rate (AGR), which has been shown to occur during osteon formation. Scanning electron microscopy (SEM) was used to count the number of canalicular openings per unit surface on large Haversian canals of forming osteons as well as on small canals of completed osteons. Reflected polarized light microscopy (RPL) enabled the number per unit length of canaliculi to be counted at different concentric levels of the osteons. The results of both techniques agree in showing that, in the subjects examined, OCD does not change significantly throughout the osteon wall. Since no correlation exists between OCD and AGR, it follows that osteoblast flattening, which was shown to occur in parallel to the decrement of the rate of concentric bone deposition, does not seem to depend primarily on the number of osteoblast-osteocyte contacts, but on other factors.


1993 - Osteoconduzione del Fosfato Tricalcico e del Corallo [Articolo su rivista]
Zaffe, Davide; Ferretti, Marzia; E., Cantoni; M., Tassinari
abstract

Granuli di Fosfato Tricalcico (TCP) o di Corallo madreporico sono stali impiantati in cavità post-estrallive di 14 Uomini adulti; dopo 3-75 mesi, in dascun soggetto è stata effettuata una biopsia, poi fissata in paraformaldeide e inclusa in PMMA, e quindi trattata per il microscopio ottico, SEM e microsonda a raggi-X. I risultati dimostrano che tra i granuli di Corallo l'osso neoformato è già presente dopo 3 mesi, mentre tra i granuli di TCP non compare prima di 6 mesi. I granuli di Corallo scompaiono dalle biopsie dopo 10-12 mesi, mentre granuli di TCP hanno dimensioni simili a quelle iniziali anche dopo 15 mesi di impianto. L'osso neodeposto, inoltre, viene assai più estesamente a diretto contatto con il Corallo che con i granuli di TCP. La microsonda ai raggi-X dimostra infine che l'osso a ridosso del Corallo ha un contenuto minerale più elevato. La neodeposizione avviene pertanto in tempi più brevi e con migliori modalità negli impianti di Corallo rispetto a quelli di TCP; la più elevala capacità riparatrice di difelli ossei mostrata dal Corallo è verosimilmente da attribuire alla maggiore reattività del Carbonato di Calcio rispetto a quella del Fosfato.The alveolar cavities, obtained after the removal of a toolh in 14 adult men, were filled with TriCalcium Phosphate (TCP) or Coral; from 3 to 75 months after the implant, a biopsy was made and processed for optical microscope, SEM and X-ray microanalysis. The results show that bone tissue is present among the Coral granules after 3 months, whereas around TCP granules bone does not appear before 6 months; Coral granules disappear from biopsies after 10-12 months, whereas TCP granules have the same size as at the start even after 75 months; there is direct bone-Coral contact but only a partial bone-TCP contact. X-ray microanalyses show that the bone mineralization is higher close to the Coral than near the TCP. In conclusion, the new bone formation appears to take place at a higher rate in Coral implants than in TCP ones; the higher capability shown by Coral in bone defect repair is probably due lo the higher reactivity of Calcium Carbonate with respect to Calcium Phosphate.


1992 - A quantitative evaluation of osteoblast-osteocyte relationships on growing endosteal surface of rabbit tibiae. [Articolo su rivista]
Marotti, Gastone; Ferretti, Marzia; Muglia, Maria Antonietta; Palumbo, Carla; S., Palazzini
abstract

Scanning electron microscopy (SEM) was used to quantify the intercellular relationships between osteoblasts and osteocytes on the growing endosteal surfaces of the medullary canal of the tibia in four rabbits of different ages. The area of each osteoblast was measured on the SEM micrographs by means of an Image Analyzer. The number of osteocyte cytoplasmic processes was indirectly evaluated by counting the canalicular openings present on the same microscopic fields after the removal of the osteoblasts. The metabolic activity of the osteoblasts was indirectly evaluated from their shape, and the structure was analyzed by transmission electron microscope (TEM) in sections taken from the samples studied by SEM. In all four animals, the surface area of the osteoblasts (OA) was found to vary a great deal, whereas the density of canalicular openings was fairly uniform. Moreover, although the OA mean value increases significantly with the age of the animals, the density of canalicular openings does not; it would therefore appear that the older the animal and the more flattened the osteoblasts, the greater the number of canaliculi beneath them. Since osteoblast activity has previously been shown to be inversely proportional to the area of the protoplasm in contact with the bone surface, it appears that the less active osteoblasts should contact a greater number of osteocyte cytoplasmic processes. These findings suggest that osteocytes might play an important role in modulating osteoblast activity and in recruiting osteoblasts that differentiate into osteocytes, possibly by means of inhibitory signals transmitted via gap junctions.


1992 - Quantitative aspects on osteoblast-osteocyte relationships. [Articolo su rivista]
Marotti, Gastone; Ferretti, Marzia; Muglia, M. A.; Palumbo, Carla
abstract

Osteocytes could modulate osteoblast activity by issuing inhibitory signals.


1991 - A SEM quantitative-study of osteoblast-osteocyte relationships on endosteal surface of growing rabbits. [Abstract in Rivista]
Marotti, Gastone; Ferretti, Marzia; Muglia, M. A.; Palumbo, Carla
abstract

The less active osteoblasts contact a greater number of osteocyte cytoplasmic processes. This fact suggest the hypothesis that osteocytes might exert an inhibitory effect on the secretory activity of osteoblasts.