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Francesco CAVANI

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

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Pubblicazioni

2024 - Piezosurgery versus Reciprocating Saw: Qualitative Comparison of the Morphology of Cutting Surfaces in Ex Vivo Human Bone [Articolo su rivista]
Anesi, Alexandre; Negrello, Sara; Checchi, Marta; Di Bartolomeo, Mattia; Salvatori, Roberta; Cavani, Francesco; Palumbo, Carla; Ferretti, Marzia
abstract

The aim of this study was to morphologically evaluate the differences in the cutting surfaces of bone segments obtained by reciprocating saw (RS) and two piezosurgical devices (Piezosurgery Medical—PM—and Piezosurgery Plus—PP) in ex vivo human fibulae. The ultimate goal was to identify the presence of debris, scratches, and microcracks on the cutting surface that might affect bone healing, a key aspect in oral and maxillofacial surgery. Ten patients who underwent a microsurgical reconstruction of the mandible with a free fibula flap were enrolled. The fibula segments usually discarded after surgery were cut using RS, PM, and PP, obtaining transverse sections to analyze under an environmental scanning electron microscope to perform a histomorphological qualitative evaluation. Bone surfaces cut with the RS presented several scratches, and haversian canals were frequently filled with bone debris/chips. On the contrary, PM and PP devices produced smoother and sharper cutting surfaces, with lower production of bone debris/chips, preventing vascular spaces’ closure. Microcracks were found in both PM and PP cut specimens, and they could be associated with the triggering of bone remodeling, thus improving the formation of new bone, while their presence was rarely observable in RS cut samples. The use of piezosurgical devices showed superior performance, providing cleaner and smoother cutting surfaces that favor vascularization and bone remodeling; altogether, these processes could lead to accelerated bone healing, a fundamental goal in all surgical procedures that involve bone cutting.

2024 - Push-out bond strength and SEM fractographical analysis of hollow fibre posts used with self-adhesive resin cement: a pilot study [Articolo su rivista]
Generali, Luigi; Veneri, Federica; Forabosco, Eleonora; Cavani, Francesco; Piergianni, Veronica; Sassatelli, Paolo; Checchi, Vittorio; Pedullà, Eugenio
abstract

Detachment is the major cause of failure of endodontic fibre posts. Hollow posts have been recently introduced to overcome such issue. The primary aim of this pilot study was to compare the push-out bond strength of hollow posts and traditional solid posts. Eight round-shaped single-canal premolars extracted for periodontal reason were selected as sample and equally randomized into two groups: (i) traditional solid fibre posts-TECH21xop and ii) hollow fibre posts-TECHOLE. A dual-curing self-adhesive cement (new TECHCEM) was used for posts placement. Six horizontal sections-two from each portion of the root (coronal, middle and apical)-were obtained from each sample root, yielding a total of 24 sections for each group. Push-out test was performed on the sections and bond strength values were compared between groups and within each group. Scanning electron microscope (SEM) fractographical analysis was conducted on each section. Additional SEM and EDX analyses were performed on new samples of both posts, to assess fibres density and distribution, and the chemical composition of the fibres and the matrix. Hollow posts showed a significantly higher push-out bond strength (6.36 +/- 1.22 MPa) than solid posts (3.64 +/- 1.62 MPa). Among the three root portions of the same group, there was no significant difference in bond strength. In both groups, the most frequent type of fracture was a mixed adhesive failure with the cement covering 0 to 50% of the post perimeter. Hollow post fibres appear more similar in size and have a more homogeneous distribution, compared to solid posts. The two post types also have different chemical compositions.

2024 - Specific subcellular localization of phosphoinositide-specific phospholipase C enzymes in different human osteosarcoma cell lines [Articolo su rivista]
Corradini, M; Checchi, M; Benincasa, M; Ferretti, M; Cavani, F; Palumbo, C; LO VASCO, VINCENZA RITA
abstract

The role of signal transduction in cancer progression is well established and actively studied, including in osteosarcoma. The signal transduction pathways involved in the regulation of calcium metabolism are being intensively studied, with particular regard to phosphoinositide-specific phospholipase C (PLC) signaling. This family of enzymes helps to modulate calcium metabolism and is interconnected with additional signaling molecules belonging to different pathways. The expression and subcellular localization of PLCs have been shown to differ in normal cells compared to their neoplastic counterpart in different types of cancer. We now describe the localization of the PLC enzyme family in 4 human osteosarcoma cells different in origin and malignancy (MG63, U2OS, HOS and 143B cell lines). We identified cell line-specific differences and discussed possible meaning and implications.

2023 - Endoplasmic reticulum localization of phosphoinositide specific phospholipase C enzymes in U73122 cultured human osteoblasts [Articolo su rivista]
Corradini, Matteo; Checchi, Marta; Ferretti, Marzia; Cavani, Francesco; Palumbo, Carla; LO VASCO, VINCENZA RITA
abstract

Different signal transduction pathways contribute to the differentiation and metabolic activities of osteoblasts, with special regard to the calcium-related pathway of phosphoinositide specific phospholipase C (PLC) enzymes family. PLC enzymes were demonstrated to be involved in the differentiation of osteoblasts, and differently localize in the nucleus, cytoplasm or both depending on the isoform. The amino-steroid molecule U-73122 inhibits the enzymes belonging to the PLC family. Beside the temporary block of the enzymatic activity, U-73122 promotes off-target effects, including modulation of the expression of selected PLC genes and different localization of PLC enzymes depending on the cell line in different cell lines. In order to evaluate possible off-target effects of the molecule in human osteoblasts, we investigated the expression of PLC genes and the localization of PLC enzymes in cultured human osteoblasts (hOBs) in the presence of low dose U-73122. Our results confirm that all PLC genes are transcribed in hOBs, that probably splicing variants of selected PLC genes are expressed, and that all PLC enzymes are present in hOBs, excepting for PLC 3 in quiescent hOBs at seeding. Our results confirm literature data excluding toxicity of U-73122 upon cell survival. Our results indicate that U-73122 did not significantly affect the transcription of PLC genes. It acts upon the localization of PLC enzymes, as PLC enzymes are detected in cell protrusions or pseudopodia-like structures, at the nuclear or the plasma membrane, in membrane ruffles, and/or in the endoplasmic reticulum.

2023 - In-vivo evaluations of bone regenerative potential of two novel bioactive glasses [Articolo su rivista]
Anesi, A.; Ferretti, M.; Salvatori, R.; Bellucci, D.; Cavani, F.; Di Bartolomeo, M.; Palumbo, C.; Cannillo, V.
abstract

: Due to the aging of population, materials able to repair damaged tissues are needed. Among others, bioactive glasses (BGs) have attracted a lot of interest due to their outstanding properties both for hard and soft tissues. Here, for the first time, two new BGs, which gave very promising results in preliminary in vitro-tests, were implanted in animals in order to evaluate their regenerative potential. The new BGs, named BGMS10 and Bio_MS and containing specific therapeutic ions, were produced in granules and implanted in rabbits' femurs for up to 60 days, to test their biocompatibility and osteoconduction. Additionally, granules of 45S5 Bioglass® were employed and used as a standard reference for comparison. The results showed that, after 30 days, the two novel BGs and 45S5 displayed a similar behavior, in terms of bone amount, thickness of new bone trabeculae and affinity index. On the contrary, after 60 days, 45S5 granules were mainly surrounded by wide and scattered bone trabeculae, separated by large amounts of soft tissue, while in BGMS10 and Bio_MS the trabeculae were thin and uniformly distributed around the BG granules. This latter scenario could be considered as more advantageous, since the features of the two novel BG granules allowed for the neo-formation of a uniformly distributed bony trabeculae, predictive of more favorable mechanical behavior, compared to the less uniform coarse trabeculae, separated by large areas of soft tissue in 45S5 granules. Thus, BGMS10 and Bio_MS could be considered suitable products for tissue regeneration in the orthopedic and dental fields.

2023 - Influence of heat treatment on fatigue resistance of two NiTi endodontic files [Articolo su rivista]
Generali, Luigi; Veneri, Federica; Barbieri, Marco; Bolelli, Giovanni; Lusvraghi, Luca; Cavani, Francesco; Matteucci, Tommaso; Pedullà, Eugenio
abstract

OBJECTIVES To evaluate the cyclic, torsional fatigue resistance and phase transformation of two heat-treated and non-heat-treated nickel-titanium reciprocating instruments. MATERIALS AND METHODS Twenty non-heat-treated (Procodile, Komet, Brasseler GmbH & Co., Lemgo, Germany) and 20 heat-treated (Procodile Q, Komet, Brasseler GmbH & Co., Lemgo, Germany) files (25 mm length, #25 apical diameter and 0.6 taper) were subjected to fatigue resistance tests. The dynamic cyclic fatigue was tested at 35 & PLUSMN;1 & DEG;C, using a dedicated patented device, in an artificial stainless-steel canal with a 60 & DEG; angle of curvature, the plate containing the artificial canal performing a controlled axial upand-down movement at 8 mm/s speed. The instruments were operated with a specific reciprocating motion (Reflex Dynamic & REG;, Komet, Breasseler GmbH & Co., Lemgo, Germany). Time to fracture (TtF) was recorded and the length of the fractured tips was measured. The torsional fatigue resistance was tested at room temperature (21 & PLUSMN;1 & DEG;C) using a custom-made device manufactured according to ISO 3630-1. The instruments were fixed 3 mm from the tip and their shafts were rotated counterclockwise at a speed of 2 rpm until fracture. The maximum torque load (Ncm) and corresponding rotation angle at fracture were recorded. All the results were statistically analysed (p <0.05). Fractographic analysis was performed using a field emission gun scanning electron microscope (FEG-SEM) to disclose the fractured surface characteristics. To assess the temperature range for phase transformations, differential scanning calorimetry (DSC) was performed on small segments of & AP;20 mg mass subjected to 2 heating and 2 cooling thermal cycles at rates of 5 & DEG;C min-1 in flowing N2 atmosphere over a temperature range of-40 & DEG;C to +110 & DEG;C. RESULTS The heat-treated instruments showed a greater resistance to dynamic cyclic fatigue as compared to the non-heat-treated sample (TtF 303 & PLUSMN;18.5 s vs 220 & PLUSMN;18.4 s; p <0.05) and a higher resistance to torsional fracture, bearing a greater maximum torque load (1.67 & PLUSMN;0.16 vs 0.82 & PLUSMN;0.07 Ncm; p <0.05). No significant differences were detected between heat-treated and non-heat-treated samples in mean angular rotation to fracture (298 & PLUSMN;25 & DEG; i,�312 & PLUSMN;32 & DEG;; p >0.05) and in the mean length of the fractured fragments (p >0.05). All instruments showed both ductile and brittle fracture patterns. According to the DSC, the direct (cooling) and reverse (heating) transformations of the non-heat-treated files occurred at lower temperatures (<25 & DEG;C) than those of the heat-treated files (& AP;50 & DEG;C), the latter thus not being austenitic at room and body temperatures at which they are operated. Also, the different transformation enthalpies suggest a multi-step transformation, likely involving R-phase formation, for heat-treat-ed files, against a direct transition between austenitic and martensi-tic phase for non-heat-treated files. CONCLUSIONS According to these findings, heat treatment of the tested files pro-vides them with microstructural properties more suited to the clin-ical operating conditions and im-proved performances in terms of torsional and flexural strength. CLINICA L SIGNIFICANCE Heat-treated files might be the best choice, over the traditional non-heat-treated files, when fac-ing challenging clinical condi-tions, such as curved and con-stricted canals.

2023 - Unexpected Absence of Skeletal Responses to Dietary Magnesium Depletion: Basis for Future Perspectives? [Articolo su rivista]
Ferretti, Marzia; Cavani, Francesco; Lo Vasco, Vincenza Rita; Checchi, Marta; Truocchio, Serena; Davalli, Pierpaola; Frassineti, Chiara; Rizzi, Federica; Palumbo, Carla
abstract

2022 - Calcium hydroxide removal using four different irrigation systems: A quantitative evaluation by scanning electron microscopy [Articolo su rivista]
Generali, L.; Cavani, F.; Franceschetti, F.; Sassatelli, P.; Giardino, L.; Pirani, C.; Iacono, F.; Bertoldi, C.; Angerame, D.; Checchi, V.; Pedulla, E.
abstract

This study compares conventional endodontic needle irrigation, passive ultrasonic irriga-tion, apical negative pressure irrigation, and mechanical activation to remove calcium hydroxide from single straight root canals. Eighty-four mandibular premolars were prepared in a crown-down manner up to size #40. Two teeth represented a negative control, and another two served as a positive control. Calcium hydroxide paste was placed inside root canals. The remaining eighty samples were analyzed based on the activation techniques, and the cleanliness of the canals was quantified using Fiji’s software on 500× magnified SEM backscattered electron micrographs. Considering the whole canal, all instruments showed better performance than conventional endodontic needle irrigation in removing calcium hydroxide (p < 0.05). Irrisafe and XP-endo Finisher could remove a significantly higher amount of calcium hydroxide than Endovac (p < 0.05). Irrisafe and XP-endo Finisher have been able to remove more calcium hydroxide than EndoVac.

2022 - Editorial for the Special Issue on “Multidisciplinary Insights on Bone Healing” [Articolo su rivista]
Anesi, A.; Cavani, F.
abstract

: Animal and human bone damage can be considered differently according to a macro- or micro-level analysis [...].

2022 - Evaluation of the root filling quality with experimental carrier-based obturators: a CLSM and FEG-SEM analysis [Articolo su rivista]
Pirani, C.; Generali, L.; Iacono, F.; Cavani, F.; Prati, C.
abstract

This study evaluated tubule penetration of GuttaFlow Bioseal with cold single cone or carrier-based technique, under confocal laser scanning microscopy (CLSM). Twenty straight single-rooted teeth were instrumented with Hyflex CM and divided in two groups (n = 10) according to the obturation method: single cold gutta-percha cones; experimental carrier-based obturators. GuttaFlow Bioseal, labelled with Rhodamine B dye, was used as sealer in both groups. Teeth transversally sectioned were observed under CLSM. Percentage of sealer penetration and integrity of sealer layer perimeter were measured. Surface and microstructural characteristics of obturators and gutta-percha cones were compared by FEG-SEM and EDX analysis. No significant differences were found between groups for each examined parameter. Significant differences (P < 0.05) were reported mainly within groups. Integrity was similar among and within groups. FEG-SEM/EDX analysis of obturators revealed the presence of Ba and Zn. Carrier-based obturation technique associated with GuttaFlow Bioseal does not seem to affect sealer penetration into dentinal tubules.

2022 - Pulsed Electro-Magnetic Field (PEMF) Effect on Bone Healing in Animal Models: A Review of Its Efficacy Related to Different Type of Damage [Articolo su rivista]
Di Bartolomeo, M.; Cavani, F.; Pellacani, A.; Grande, A.; Salvatori, R.; Chiarini, L.; Nocini, R.; Anesi, A.
abstract

Biophysical energies are a versatile tool to stimulate tissues by generating biopotentials. In particular, pulsed electromagnetic field (PEMF) stimulation has intrigued researchers since the 1970s. To date, many investigations have been carried out in vivo, but a gold standard treatment protocol has not yet been defined. The main obstacles are represented by the complex setting of PEMF characteristics, the variety of animal models (including direct and indirect bone damage) and the lack of a complete understanding of the molecular pathways involved. In the present review the main studies about PEMF stimulation in animal models with bone impairment were reviewed. PEMF signal characteristics were investigated, as well as their effect on molecular pathways and osseous morphological features. We believe that this review might be a useful starting point for a prospective study in a clinical setting. Consistent evidence from the literature suggests a potential beneficial role of PEMF in clinical practice. Nevertheless, the wide variability of selected parameters (frequency, duration, and amplitude) and the heterogeneity of applied protocols make it difficult to draw certain conclusions about PEMF effectiveness in clinical implementation to promote bone healing. Deepening the knowledge regarding the most consistent results reported in literature to date, we believe that this review may be a useful starting point to propose standardized experimental guidelines. This might provide a solid base for further controlled trials, to investigate PEMF efficacy in bone damage conditions during routine clinical practice.

2021 - Comparative evaluation of the penetration depth into dentinal tubules of three endodontic irrigants [Articolo su rivista]
Giardino, L.; Pedullà, E.; Cavani, F.; Bisciotti, F.; Giannetti, L.; Checchi, V.; Angerame, D.; Consolo, U.; Generali, L.
abstract

This study aimed to examine the penetration depth into dentinal tubules of some chelating agents. The 17% EDTA and two preparations containing surfactants (Smear Clear, Bioakt Endo) were tested. Surface tension and liquid viscosity were measured using a Dynamic Contact Angle Analyzer and a Haake rotational rheometer. To measure the penetration depth inside dentinal tubules, thirty maxillary central incisors were selected from a pool of extracted human permanent teeth and allocated to three experimental groups (10 samples each), as well as were mechanically shaped and cleansed with 5.25% NaOCl, followed by each of the chelators being labeled with 0.1 wt % Rhodamine B according to final irrigation protocol established. The samples were embedded in an epoxy resin, after which 200 μm thick transverse sections were obtained at 2, 5, and 8 mm from the apex with a saw microtome. The specimens were then observed using a confocal laser microscope (CLSM) and the penetration of the labeled solution was measured in every third of each sample. Statistical analysis was performed using ANOVA or Kruskal–Wallis tests according to the distribution of data, evaluated with the Shapiro–Wilk normality test. Viscosity and surface tension tests have shown that BioAKT Endo has the lowest values compared to EDTA and Smear Clear. The medium penetration depth did not significantly differ among the three irrigants, while it increased considerably from the apical to the coronal level in all groups. Additionally, the maximum penetration depth increased significantly from the apical to coronal level, while among groups, BioAKT Endo showed the highest values at the apical and middle level compared to the other irrigants. No significant differences were observed among the three groups in medium and maximum penetration depths when the entire root was considered. New irrigants containing surfactants show reduced surface tension and, in one case (BioAKT Endo), viscosity. The lowering of the surface tension allows for better penetration of liquids into dentinal tubules than EDTA alone, thus improving the cleaning of the root canal system.

2021 - Identification of Sclerostin as a Putative New Myokine Involved in the Muscle-to-Bone Crosstalk [Articolo su rivista]
Magarò, Maria Sara; Bertacchini, Jessika; Florio, Francesca; Zavatti, Manuela; Potì, Francesco; Cavani, Francesco; Amore, Emanuela; De Santis, Ilaria; Bevilacqua, Alessandro; Reggiani Bonetti, Luca; Torricelli, Pietro; Maurel, Delphine B.; Biressi, Stefano; Palumbo, Carla
abstract

Bone and muscle have been recognized as endocrine organs since they produce and secrete “hormone-like factors” that can mutually influence each other and other tissues, giving rise to a “bone–muscle crosstalk”. In our study, we made use of myogenic (C2C12 cells) and osteogenic (2T3 cells) cell lines to investigate the effects of muscle cell-produced factors on the maturation process of osteoblasts. We found that the myogenic medium has inhibitory effects on bone cell differentiation and we identified sclerostin as one of the myokines produced by muscle cells. Sclerostin is a secreted glycoprotein reportedly expressed by bone/cartilage cells and is considered a negative regulator of bone growth due to its role as an antagonist of the Wnt/β-catenin pathway. Given the inhibitory role of sclerostin in bone, we analyzed its expression by muscle cells and how it affects bone formation and homeostasis. Firstly, we characterized and quantified sclerostin synthesis by a myoblast cell line (C2C12) and by murine primary muscle cells by Western blotting, real-time PCR, immunofluorescence, and ELISA assay. Next, we investigated in vivo production of sclerostin in distinct muscle groups with different metabolic and mechanical loading characteristics. This analysis was done in mice of different ages (6 weeks, 5 and 18 months after birth) and revealed that sclerostin expression is dynamically modulated in a muscle-specific way during the lifespan. Finally, we transiently expressed sclerostin in the hind limb muscles of young mice (2 weeks of age) via in vivo electro-transfer of a plasmid containing the SOST gene in order to investigate the effects of muscle-specific overproduction of the protein. Our data disclosed an inhibitory role of the muscular sclerostin on the bones adjacent to the electroporated muscles. This observation suggests that sclerostin released by skeletal muscle might synergistically interact with osseous sclerostin and potentiate negative regulation of osteogenesis possibly by acting in a paracrine/local fashion. Our data point out a role for muscle as a new source of sclerostin.

2021 - Three-Punch Alveolar Ridge Reconstruction Technique: A Novel Flapless Approach in Eight Consecutive Cases [Articolo su rivista]
Grassi, A; Bernardello, F; Cavani, F; Palumbo, C; Spinato, S
abstract

This clinical and histologic case series aims to evaluate a novel flapless approach to alveolar ridge reconstruction (ARR) of compromised extraction sockets by means of collagenated xenograft sealed with three resorbable layers of hole punched membrane. Eight postextraction sockets without buccal and/or palatal bone walls and with adjacent natural teeth from eight consecutive patients were included. Pretreatment CBCT scanning was performed. After debridement of the selected sites, a flapless grafting procedure was carried out, and the three membrane protection was applied. After 6 to 17 months, at implant placement, a posttreatment alveolar ridge CBCT was taken, and a bone core biopsy sample was harvested for histologic and morphometric analyses. Clinical outcomes showed predictable horizontal bone regeneration in all postextraction sockets with good preservation of soft tissue architecture. Pretreatment ridge CBCT measurements showed limited bone width (2.6 +/- 1.08 mm). Posttreatment measurements revealed adequate bone width (9.05 +/- 1.29 mm) with a mean bone gain of 6.4 +/- 1.34 mm. Histologic and morphometric analyses revealed the absence of inflammatory cells and the presence of 25.4% +/- 8.7% of new bone and 31.8% +/- 8.3% of graft particles inside the biopsy samples. Many graft particles were surrounded and interconnected by new bone, thus demonstrating the formation of a bone-graft network. Rare osteoclasts were found. This novel technique seems to be effective in treating alveolar sockets prior to implant placement, preventing inflammation and bone resorption and promoting bone regeneration. Int J Periodontics Restorative Dent 2021;41:875-884. doi: 10.11607/prd.4913

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 - Scleral ossicles: Angiogenic scaffolds, a novel biomaterial for regenerative medicine applications [Articolo su rivista]
Checchi, M.; Bertacchini, J.; Cavani, F.; Magaro, M. S.; Reggiani Bonetti, L.; Pugliese, G. R.; Tamma, R.; Ribatti, D.; Maurel, D. B.; Palumbo, C.
abstract

Given the current prolonged life expectancy, various pathologies affect increasingly the aging subjects. Regarding the musculoskeletal apparatus, bone fragility induces more susceptibility to fractures, often not accompanied by good ability of self-repairing, in particular when critical-size defects (CSD) occur. Currently orthopedic surgery makes use of allografting and autografting which, however, have limitations due to the scarce amount of tissue that can be taken from the donor, the possibility of disease transmission and donor site morbidity. The need to develop new solutions has pushed the field of tissue engineering (TE) research to study new scaffolds to be functionalized in order to obtain constructs capable of promoting tissue regeneration and achieve stable bone recovery over time. This investigation focuses on the most important aspect related to bone tissue regeneration: the angiogenic properties of the scaffold to be used. As an innovative solution, scleral ossicles (SOs), previously characterized as natural, biocompatible and spontaneously decellularized scaffolds used for bone repair, were tested for angiogenic potential and biocompatibility. To reach this purpose, in ovo Chorioallantoic Membrane Assay (CAM) was firstly used to test the angiogenic potential; secondly, in vivo subcutaneous implantation of SOs (in a rat model) was performed in order to assess the biocompatibility and the inflammatory response. Finally, thanks to the analysis of mass spectrometry (LCMSQE), the putative proteins responsible for the SO angiogenic properties were identified. Thus, a novel natural biomaterial is proposed, which is (i) able to induce an angiogenic response in vivo by subcutaneous implantation in a non-immunodeficient animal model, (ii) which does not induce any inflammatory response, and (iii) is useful for regenerative medicine application for the healing of bone CSD.

2020 - Volumetric Changes Following Lateral Guided Bone Regeneration [Articolo su rivista]
Mordini, L.; Hur, Y.; Ogata, Y.; Finkelman, M.; Cavani, F.; Steffensen, B.
abstract

Resorbable membranes are well described and employed for horizontal guided bone regeneration (GBR). However, the currently available literature does not provide information on the bone volumetric changes during the healing that follows GBR procedures and dental implant placement. Therefore, the aim of this pilot study was to initially analyze the volumetric bone changes after treating pristine edentulous mandibular defects with lateral GBR using freeze-dried bone allograft (FDBA) and collagen resorbable membrane. Six patients were selected for the analysis. Clinical changes in bone volume before and after GBR were measured. In addition, digital volumetric analysis of the augmented ridges was performed preoperatively, as well as 4 and 6 months after the GBR procedure. At the time of dental implant placement, bone cores were collected during the osteotomy for histologic analysis. Data on volume changes showed a mean of 297.5 ± 134 mm3 augmented bone volume at 4 months with 5% ± 3.78% resorption from 4 to ≥ 6 months. Histologic bone core analysis showed 44.9% plusmn; 5.1% mineralization in the area of augmentation. Within the limitations of this pilot study, resorbable membranes exhibited reliability for GBR in intercalated mandibular defects, providing sufficient bone volume gain at ≥ 6 months for implant stabilization and limited resorption during graft healing.

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 - 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 - Effect of Self-Adjusting file and WaveOne reciprocating file on the filling ability of oval-shaped canals with thermoplasticized gutta-percha [Articolo su rivista]
Generali, L; Cavani, F; Righi, E; Murri Dello Diago, A; Spinas, E; Giannetti, L
abstract

The aim of the study was to compare the effect of Self-Adjusting Files (SAF) and WaveOne Primary file with syringe and needle irrigation on the filling ability of oval-shaped root canals obturated with thermoplasticized gutta-percha. Twenty-four single root teeth with single oval-shaped root canals were distributed in two experimental homogeneous group. One group was instrumented and cleansed using the SAF system while in the other group the WaveOne system with syringe and needle irrigation was used. After instrumentation, the roots were filled by Thermafil Obturators and TopSeal sealer. Specimens were transversally sectioned at 2-, 5- and 7-mm levels from the apex and observed under light microscope. The percentage of gutta-percha filled area (PGFA), the percentage of sealer filled area (PSFA) and the percentage of voids area (PVA) were measured for each section, moreover the percentage of completely filled sections was evaluated. At all levels, no significant differences in terms of PGFA, PSFA, PVA and percentage of completely filled canals between groups were obtained (P > .05). On the contrary, when the data were pooled, the mean PGFA in the SAF group was 95.8%, whereas it was 93.2% in the WaveOne group (P < 0.05). The percentage of sections completely filled was 77.8% in the SAF group, while 52.8% in the WaveOne group (P < 0.05). Overall, the use of the SAF system in oval canals allows to obtain a significantly greater complete filling than the use of the WaveOne system.

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 - Plaque accumulation on titanium disks with different surface treatments: an in vivo investigation [Articolo su rivista]
Conserva, Enrico; Generali, Luigi; Bandieri, Alberto; Cavani, Francesco; Borghi, Francesco; Consolo, Ugo
abstract

Implants with rough surfaces are today widely used. It has been speculated that rough surfaces (Ra [ 0.2 lm) provide a better ‘‘substrate’’ for retention and accumulation of plaque in terms of area, thickness and colony- forming unit that can eventually lead to peri mucositis and/or peri-implantitis. The aim of this investigation was to evaluate in vivo the plaque accumulation after 48 h on three implant surfaces with different treatments. For this investigation, we used 21 sterilized titanium disks, with a diameter of 8mm and a thickness of 3 mm, provided by the manufacturer: 7 with machined surface, as smooth control, 7 with HA grit sand- blasted RBM surface and 7 with Ca?? incorporated in tita- nium Xpeed surface. One disk for each surface treatment was characterized at time 0 by SEM and AFM to study, respec- tively, the surface morphology and roughness. The other 18 disks were mounted randomly on three upper acrylic bites in a buccal lateral position, worn for 48 h by three volunteer stu- dents for plaque accumulation. After 48 h each disk was removed and analyzed qualitatively and quantitatively by an independent operator, not involved into the study, in order to avoid bias. Data collected were statistically analyzed by one- way ANOVA. The qualitative analysis showed no differences in terms of total plaque accumulation between the surfaces. Data from quantitative analysis using Anova Test showed no significance between all groups. In this in vivo investigation all the surfaces studied promoted plaque formation. The degree of surface roughness seems not to be a critical factor for plaque accumulation.

2018 - Sodium hypochlorite penetration into dentinal tubules after manual dynamic agitation and ultrasonic activation: a histochemical evaluation [Articolo su rivista]
Generali, Luigi; Campolongo, Erica; Consolo, Ugo; Bertoldi, Carlo; Giardino, Luciano; Cavani, Francesco
abstract

The aim of this study was to compare the effects of Manual Dynamic Agitation and Passive Ultrasonic Irrigation on sodium hypochlorite (NaOCl) penetration into dentinal tubules using its bleaching ability. Thirty-four single-rooted teeth with round- shaped root canals were distributed in two homogeneous groups and one control group, characterized by different NaOCl activation systems: Manual Dynamic Agitation and Passive Ultrasonic Irrigation. After instrumentation, all root canals were stained with 10% copper sulphate solution followed by 1% rubeanic acid alcohol solution under vacuum. Final irrigation was performed with 5 mL of 5.25% NaOCl solution for 1 min and activated with Manual Dynamic Agitation or Passive Ultrasonic Irrigation for another 1 min depending on the treatment group. The teeth were transversely sectioned at the middle portion of the apical, middle, and coronal thirds and observed under light microscope. NaOCl solution penetration was evaluated by measuring the percentage of bleached circumference of the root canal relative to the stained circumference, bleached areas, mean, and maximum penetration depth. No differences in the evaluated parameters were observed between groups (p &gt; 0.05). Within groups, an increase of values was recorded from apical to coronal direction as for percentage of staining, percentage of bleaching and bleached area. NaOCl penetration into dentinal tubules did not significantly vary among the three levels. No significant differences in penetration of sodium hypochlorite into dentinal tubules when activated by means of Manual Dynamic Agitation or Passive Ultrasonic Irrigation were observed in the apical, middle, and coronal thirds of teeth with single straight round root canals.

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 - 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 - Double dye technique and fluid filtration test to evaluate early sealing ability of an endodontic sealer [Articolo su rivista]
Generali, Luigi; Prati, C; Pirani, Chiara; Cavani, Francesco; Gatto, M. R; Gandolfi, M. G.
abstract

Objectives The aim of the present study is to evaluate the possible correlation between sealer penetration into dentinal tubules and sealing ability both in presence and absence of smear layer. Materials and methods Fourteen maxillary central incisors were treated with 5.25 % NaOCl +10 % EDTA to remove the smear layer (SL-free group) or 5.25 % NaOCl without EDTA (SL group). Root canals were filled using #25 Thermafil Obturators with Topseal sealer labelled with 0.1 wt% rhodamine B. Sealing ability was measured as fluid filtration rate with a fluid-flow meter using water supplemented with 0.3 % calcein fluorescent dye. Specimens were sectioned, observed under confocal microscope to co-localize the presence of sealer (rhodamine B labelling) into dentinal tubules and gaps (calcein labelling) into the root canal. The depth of sealer penetration into dentinal tubules and the percentage of sealer penetration around the root canal were measured at 3, 5 and 8 mm from the apex. Results No significant differences between groups were observed in fluid filtration rate nor in depth of calcein penetration. Sealer penetration depth and percentage into dentinal tubules were not significantly different between groups, except at 8-mm level in absence of smear layer. Conclusion Sealer penetration at 3- and 5-mm levels was not influenced by smear layer while it was significantly reduced at 8-mm level. Fluid filtration rate was not correlated either with depth of calcein penetration nor with sealer penetration into dentinal tubules.

2017 - Effect of Different Irrigation Systems on Sealer Penetration into Dentinal Tubules [Articolo su rivista]
Generali, Luigi; Cavani, Francesco; Serena, Valentina; Pettenati, Corinne; Righi, Elena; Bertoldi, Carlo
abstract

Introduction: Different irrigation systems have been developed to improve the efficacy and distribution of the irrigants. The aim of this study was to compare the effect of conventional endodontic needle irrigation with other irrigant delivery and/or agitation systems on sealer penetration into dentinal tubules. Methods: Fifty single-rooted teeth with round-shaped root canals were distributed in 5 homogeneous groups character- ized by the different cleansing system used: conven- tional endodontic needle irrigation, EndoActivator, Irrisafe, Self-Adjusting File, and EndoVac. After instru- mentation, all teeth were filled by Thermafil obturators and rhodamine B dye labeled TopSeal sealer. Teeth were transversally sectioned at 2-, 5-, and 7-mm levels from the apex and observed under confocal laser scanning microscope. Maximum, mean, and percentage of sealer penetration inside tubules around the root canal were measured. Moreover, the integrity of the sealer layer perimeter was evaluated. Results: No significant differ- ences both in mean (p &gt; .05) and in maximum penetra- tion depth (p &gt; .05) were observed among groups, whereas both parameters showed an increased trend within each group from the 2- to the 7-mm level from apex. Similarly, the percentage of penetration around the root canal wall did not differ among groups (p &gt; .05) and showed an increasing trend within each group from the apical to the coronal portion of the canal. Conclusions: Sealer penetration into dentinal tubules is not affected by the irrigant delivery and/or agitation sys- tems studied. Thermafil with TopSeal technique achieves complete sealer perimeter integrity in all groups.

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.&nbsp;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 - Histochemical evaluation of activated sodium hypochlorite into human dentin [Abstract in Rivista]
Generali, Luigi; Cavani, Francesco; Campolongo, Erica; Bertoldi, Carlo; Consolo, Ugo
abstract

Aim: The aim of this study was to evaluate ex vivo the penetration depth of sodium hypochlorite in human dentin, with manual and ultrasonic activation, using a histochemical staining technique under vacuum and the bleaching ability of NaOCl. Methods: The root canals of 36 extracted single canal teeth were shaped to a size 30. .07 taper. Dentinal tubules were stained with 8% copper sulphate solution and 5% ammonium hydroxide followed by a 1% rubeanic acid alcohol solution under vacuum. The samples were irrigated for 1 min with 5mL of 5.25% sodium hypochlorite solution, then the irrigant was activated for 1 minute by Manual Dynamic Agitation (MAD) with K-file (Kerr, Italy) or Passive Ultrasonic Activation (PUI) with Irrisafe insert (Satelec Acteon group, Merignac, France), according to the corresponding treatment group. A cross-section was obtained in the central portion of each root third. Under microscopic examination, sodium hypochlorite penetration into dentin was evaluated by measuring the following parameters: bleached circumference of the root canal with respect to the stained circumference; bleached areas, mean and maximum penetration depth. For statistical analysis, Pearson’s chi-squared test and one-way ANOVA were used. Results: Only 58% of the apical sections showed stained dentinal tubules; therefore, these were excluded from further comparisons. The percentage of copper sulphate-stained circumference of the root canals increased from the middle to coronal level in both groups, with a statistically significant difference (P<0.001). In the middle and coronal third, the average root canal bleached circumference was 98-100% of the stained circumference, with no difference within and between groups, and the bleached area was 125490-298290 μm², respectively. The bleached circumference values (also those related to root canal) and bleaching areas of the coronal third were significantly higher than those recorded in the middle third in each group, while no differences between groups at the same level were observed. The mean penetration depth ranged from 63 to 69 μm and the maximum was from 96 to 106 μm, without inter- and intra-group differences (P = 1). Conclusion: No differences in dentinal tubules penetration of 5.25% sodium hypochlorite solution were observed with manual and ultrasonic activation in the middle and coronal root thirds of single canal teeth.

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&nbsp;days after surgery). The observations reported here need to be framed in the context of our previous investigations regarding bone histogenesis (Ferretti et&nbsp;al. Anat Embryol. 2002;&nbsp;206:&nbsp;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&nbsp;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.

2017 - Sodium hypochlorite solution penetration into human dentine: a histochemical evaluation [Articolo su rivista]
Giardino, L.; Cavani, Francesco; Generali, Luigi
abstract

Aim To evaluate ex vivo the penetration depth of sodium hypochlorite solutions, with and without surfactants, into human dentine using a histochemical stain and the bleaching ability of NaOCl. Methodology Extracted maxillary central incisors were decoronated and their root canals shaped to a size 30, .07 taper. Dentinal tubules were stained with 10% copper sulphate solution followed by a 1% rubeanic acid alcohol solution under vacuum. Specimens were irrigated with 5 mL of Niclor 5 (5.25% NaOCl solution) or 5 mL of Hypoclean (5.25% NaOCl solution + surfactants) according to the corresponding treatment group, for 2 minutes. A transverse section was obtained in the middle portion of the apical, middle and coronal third of each tooth. Under microscopic examination irrigant penetration was evaluated by measuring: the bleached circumference of the root canal with respect to the stained circumference; bleached areas, mean and maximum penetration depth. For statistical analysis, Pearson Chi-squared test and one-way ANOVA were used. Results Only 30% of dentine tubules in the apical portions were stained, therefore these were excluded from further comparisons. In the middle and coronal thirds the average bleached circumference of the root canal was 80-99% of the stained circumference, the bleached area was 73757 - 135107 μm2. The average penetration depth ranged from 39 to 62 μm and maximum penetration from 74 to 131 μm. For all reported results no differences within and between groups were found (P = 1). Conclusion No differences in penetration of irrigants with and without surfactants into dentinal tubules were found in middle and coronal third of incisor teeth.

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 - Efficacy of three different irrigation systems on calcium hydroxide removal from round and oval canals: a scanning electron microscopy study [Abstract in Rivista]
Generali, Luigi; Franceschetti, F.; Cavani, Francesco; Pettenati, C. .; Bellini, Pierantonio; Conserva, Enrico; Consolo, Ugo
abstract

BACKGROUND: To evaluate by means of scanning electron microscopy the effectiveness of different irrigation systems in the removal of calcium hydroxide from round and oval canals. METHODS: 76 extracted single-rooted teeth were selected. Criteria for tooth selection included one single root canal; no visible root caries, fractures, or cracks; and a completely formed apex under light microscope examination (10x). Radiographs in bucco-lingual and mesio-distal direction were taken to cal- culate the ratio between canal diameters measured in the two projections. If ratio was less than or equal to 2.5, the canal was considered round, otherwise was considered oval. According to ratio and root length teeth were divided into 8 groups, with 2 control samples. After access cavity preparation, working length was determined 1 mm short of the length where the file extruded the apical foramen. To simulate clinical conditions the apex was sealed with glue. Glide path and canal instrumentation were performed with Path File size 13, 16, 19 and ProTaper Universal rotary system in a crown-down sequence to size 40 at the WL. A size 25 lentulo spiral was used to fill teeth with calci- um hydroxide. Radiographs were taken to confirm the complete filling of the canals. Samples were kept in an incubator at 37° C for a week. Canals were cleaned with Irrisafe, Endovac, Xp Endo Finisher and conventional endodontic needle irrigation. Teeth were split along their long axis in a bucco-lingual direc- tion into 2 halves. For scanning electron microscopic analysis, tooth halves were fixed on aluminum stubs, gold sputter coated and observed with a scanning electron microscope. Apical, middle, and coronal thirds were established by dividing the roots according to their length. The center of each third was examined at 500x magnification. Images were taken using backscattered mode and analyzed with a digital processing software (ImageJ v. 1.49, National Institutes of Health, NIH, USA) for a quantitative analysis. The efficacy of the irriga- tion system was evaluated calculating the amount of calcium hydroxide left inside the canal, expressed as percentage of the total area of the image. To compare the 8 groups, the results were statistically analyzed using the analysis of variance test (ANOVA) with Bonferroni test or T-test (P<0.05). RESULTS: No statistically significant differences were observed among different thirds for each group. Comparing canal shape for each instrument, significant differences were found only for Endovac, where coronal and middle thirds of oval canals were cleaner than round ones. Analyzing the whole canal, significant difference can be found between irrigation systems and conventional irrigation in the oval ones. In round canals Irrisafe and Xp Endo Finisher are more effective compared to conventional irrigation, but only Irrisafe is more effective compared to Endovac. No statistical- ly significant differences can be observed between Endovac and conventional irrigation. CONCLUSIONS: None of the investigated techniques removed the calcium hydroxide completely. However, the results demonstrate greater removal of calcium hydroxide if the irrigant solutions are activated, compared with conven- tional endodontic irrigation. Therefore, it is recommended to activate the irrigant solutions to enhance chemical debride- ment of the root canal system.

2016 - Feasibility of Electroporation in Bone and in the Surrounding Clinically Relevant Structures: A Preclinical Investigation [Articolo su rivista]
Tschon, M.; Salamanna, F.; Ronchetti, M.; Cavani, F.; Gasbarrini, A.; Boriani, S.; Fini, M.
abstract

Skeletal metastases are a common cause of severe morbidity, reduction in quality of life and often early mortality. Consequently, improvements in therapies are necessary. Electroporation uses electric energy to alter cancer cell membrane permeability and enhance the local uptake of chemotherapeutics, thus leading to local tumor control. The aim of this study was to investigate the feasibility and safety of delivering electric field protocols causing electroporation of healthy bone and structures of clinical relevance using small and large animal models. Reversible electroporation was used in the rabbit sciatic nerve by applying 2 series of 8 pulses 100ms long at 1000 V/cm. Irreversible electroporation was used in rabbit distal femur condyles and in sheep vertebral body by applying 120 pulses 100ms long at 1750 V/cm. Any effect on surrounding sensitive structures was investigated. Reversible electroporation of sciatic nerve was associated with transient foot functional deficit that completely recovered at 30 days. Irreversible electroporation removed cells from trabeculae in the femurs of rabbits and in the vertebral body of sheep. After irreversible protocol, histology and microtomography demonstrated that the trabecular structure was maintained, the presence of new bone marrow cells, osteoblasts, and mineral apposition characterized by new trabeculae thinner than controls (P =.005) and a significant reduction in the ablated areas (−225%, P =.0219). Spinal cord, vertebral pedicles and spinal nerves showed transient edema in the absence of functional or structural alterations. Collectively, these results show that electroporation can be safely applied to bone even in the proximity of neuronal structures.

2016 - Influence of different irrigation techniques on sealer penetration into dentinal tubules: a confocal laser scanning microscope analysis [Abstract in Rivista]
Generali, Luigi; Serena, V; Cavani, Francesco; Righi, Elena; Pettenati, C; Bellini, Pierantonio; Consolo, Ugo
abstract

BACKGROUND: The instrumentation of roots canals pro- duces smear layer, which consists of organic and inorganic material and may also contain bacteria and they by-products. This layer covers the root canal walls, could obliterate den- tinal tubules and consequently can inhibit the penetration of irrigants and endodontic sealers. To obtain a good seal is necessary to use an endodontic sealer in association with a core filling material in order to fill any voids, morphological irregularities, apical deltas and dentinal tubules and to create an obturation as much as possible impervious. The aim of the study was to compare the effect of conventional endodontic needle irrigation and different irrigation systems on sealer penetration into dentinal tubules. METHODS: 40 single round root canals were selected and divided into four groups (n=10) before root canal preparation according to the final irrigation protocol: group 1 (conventional endodontic needle irrigation), group 2 (EndoActivator) group 3 (Irrisafe) group 4 (EndoVac). The root canal were shaped using Path File and ProTaper Universal rotary system in a crown-down sequence to size #40 at the WL. Obturation of the teeth was done with Thermafil Obturators size 40 with TopSeal sealer labeled with 0,1% wt Rhodamin B dye. Transverse sec- tion at 2-, 5- and 7-mm from the apex were analyzed by means of confocal laser scanning microscopy. Percentage of pen- etration around the root canal and the mean penetration into dentinal tubules at eight standardized points were calculated. RESULTS: No significant differences in percentage of pen- etration around the root canal and mean penetration depth into dentinal tubules were observed among groups when same levels were compared. Within each group both variables are significantly higher at the coronal and middle level with respect to the apical one. CONCLUSIONS: The use of the techniques employed in the present study did not significantly improve the sealer pen- etration into dentinal tubules compared with conventional endodontic needle irrigation.

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

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.

2014 - Differential efficacy of endodontic obturation procedures: an ex vivo study [Articolo su rivista]
Ardizzoni, Andrea; Generali, Luigi; Righi, Elena; Baschieri, MARIA CRISTINA; Cavani, Francesco; Manca, Lidia; Lugli, Eleonora; Migliarese, Luigi; Blasi, Elisabetta; Neglia, Rachele Giovanna
abstract

By means of a double-chamber model, different root canal filling materials and procedures were compared. Briefly, the root canals of single-rooted human teeth, extracted for periodontal reasons, were instrumented and obturated by gutta-percha/Pulp Canal Sealer EWT (PCS) or by Resilon, in association with different sealers (Real Seal, RelyX Unicem or Meta). Obturation was achieved by traditional continuous wave of condensation technique (TCWCT), a modified version of it (MCWCT), or single cone technique (SCT). The obturated roots, inserted in a double-chamber model, were sterilized by gamma irradiation. Next, Enterococcus faecalis was added to the upper chamber and the specimens were incubated at 37 °C for up to 120 days; the development of turbidity in the lower chambers' broths indicated bacterial leakage through the obturated root canals. The kinetics of leakage were analyzed in different groups by means of Kaplan-Meier statistics and compared by log-rank test. The results showed that root canals obturated with either gutta-percha/PCS using the MCWCT, Resilon/Real Seal SCT or Resilon/RelyX Unicem using the TCWCT displayed significantly better performance than the remaining groups (p &lt; 0.01). Histological evaluation, performed to investigate microbial localization inside specimens, confirmed that this parameter varied according to the obturation procedures and materials employed. This ex vivo study indicates that gutta-percha/PCS, if used with the MCWCT, is as effective as Resilon when coupled to Real Seal with the SCT or, interestingly, to RelyX Unicem with the TCWCT. These data suggest that further improvement of the currently employed root canal filling procedures is achievable, depending on both the filling materials and the technique employed, thus encouraging clinical studies in this direction.

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 - Effect of electric current stimulation in combination with external fixator on bone healing in a sheep fracture model [Articolo su rivista]
Muttini, Aurelio; Abate, Michele; Bernabò, Nicola; Cavani, Francesco; Mingozzi, Rossella; Tosi, Umberto; Cadossi, Matteo; Setti, Stefania; Giannini, Sandro; Leone, Rocco; Valbonetti, Luca
abstract

Biophysical stimulations with electric and electromagnetic fields have been demonstrated to accelerate the bone-healing rate. This study has been designed to investigate the effects of electricity directly connected with the central pins of an external fixator in an experimental osteotomy model in sheep. Thirty mg/kg of tetracycline chloride were administered on the 30th and on the 45th day after surgery for histomorphometric studies. Plain radiographs were obtained in standard projections every 15 days after surgery and were analyzed with a software program (Corel Photo-Paint Pro X2, Corel Corporation, Ottawa, Canada). The specimens obtained after 60 days were examined with histological analysis. The results show that biophysical treatment with alternating electricity in combination with external fixator enhances new-bone formation. The translational value of this study, due to the similarities between ovine and human species, suggests that this treatment could be useful in speeding the bone-healing rate both in animals and humans.

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 - In vivo effect of two different pulsed electromagnetic field frequencies on osteoarthritis [Articolo su rivista]
Veronesi, F; Torricelli, P; Giavaresi, G; Sartori, M; Cavani, Francesco; Setti, S; Cadossi, M; Ongaro, A; Fini, M.
abstract

Osteoarthritis (OA) is a joint pathology characterized by fibrillation, reduced cartilage thickness and subchondral bone sclerosis. There is evidence that pulsed electromagnetic fields (PEMFs) counteract OA progression, but the effect of two different PEMF frequencies has not yet been shown. The aim of this study was to test the effectiveness of PEMFs at two different frequencies (37 and 75 Hz) in a late OA stage in 21-month-old Guinea pigs. After 3 months of 6 h/day PEMF stimulation, histological and histomorphometric analyses of the knees were performed. At both frequencies, PEMFs significantly reduced histological cartilage score, fibrillation index (FI), subchondral bone thickness (SBT) and trabecular number (Tb.N) and increased trabecular thickness (Tb.Th) and separation (Tb.Sp) in comparison to the not treated SHAM group. However, PEMFs at 75 Hz produced significantly more beneficial effects on the histological score and FI than 37 Hz PEMFs. At 75 Hz, PEMFs counteracted cartilage thinning as demonstrated by a significantly higher cartilage thickness values than either those of the SHAM or 37 Hz PEMF-treated groups. Although in severe OA both PEMF frequencies were able to limit its progression, 75 Hz PEMF stimulation achieved the better results.

2014 - Morphological and quantitative analysis of BCL6 expression in human colorectal carcinogenesis. [Articolo su rivista]
Sena, Paola; Mariani, Francesco; Benincasa, Marta; PONZ DE LEON, Maurizio; Di Gregorio, C; Mancini, Stefano; Cavani, Francesco; Smargiassi, Alberto; Palumbo, Carla; Roncucci, Luca
abstract

The aim of the present study was to determine whether BCL6 is expressed during malignant transformation of the large bowel and to assess whether, and to what extent, immunoreactivity is related to the different stages of neoplastic progression. Samples of normal colorectal mucosa (n=22), microadenomas (n=22) and colorectal cancer (n=22), were analyzed by immunohistochemistry, immunofluorescence coupled with confocal microscopy and western blotting. Our results clearly outlined the marked increase occurring in both intensity and density of BCL6 protein expression in the normal mucosa-microadenoma-carcinoma sequence. Immunohistochemistry and immunofluorescence analyses showed that BCL6 is expressed at low levels in normal mucosa and increases in microadenoma and in cancer with statistical significance. These results were confirmed by western blotting data. The increasing expression of BCL6 in human colorectal cancer development suggests the involvement of BCL6 in tumor progression, from the earliest stages of carcinogenesis with significant increase in cancer. The enhanced understanding of the biological role of BCL6, previously shown to exert a key role in lymphomagenesis, may lead to a re-evaluation of this protein and may highlight the importance of performing further studies in order to identify novel therapeutic targets for colorectal cancer.

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 - The biocompatibility of porous vs non-porous bone cements: A new methodological approach [Articolo su rivista]
Dall'Oca, C; Maluta, T; Cavani, Francesco; Morbioli, Gp; Bernardi, P; Sbarbati, A; Degl'Innocenti, D; Magnan, B.
abstract

Composite cements have been shown to be biocompatible, bioactive, with good mechanical properties and capability to bind to the bone. Despite these interesting characteristic, in vivo studies on animal models are still incomplete and ultrastructural data are lacking. The acquisition of new ultrastructural data is hampered by uncertainties in the methods of preparation of histological samples due to the use of resins that melt methacrylate present in bone cement composition. A new porous acrylic cement composed of polymethylmetacrylate (PMMA) and β-tricalciumphosphate (β-TCP) was developed and tested on an animal model. The cement was implanted in femurs of 8 New Zealand White rabbits, which were observed for 8 weeks before their sacrifice. Histological samples were prepared with an infiltration process of LR white resin and then the specimens were studied by X-rays, histology and scanning electron microscopy (SEM). As a control, an acrylic standard cement, commonly used in clinical procedures, was chosen. Radiographic ultrastructural and histological exams have allowed finding an excellent biocompatibility of the new porous cement. The high degree of osteointegration was demonstrated by growth of neo-created bone tissue inside the cement sample. Local or systemic toxicity signs were not detected. The present work shows that the proposed procedure for the evaluation of biocompatibility, based on the use of LR white resin allows to make a thorough and objective assessment of the biocompatibility of porous and non-porous bone cements.

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.

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 - 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 - 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 - 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 - 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 - Ablation of bone cells by electroporation [Articolo su rivista]
Fini, M; Tschon, M; Ronchetti, M; Cavani, Francesco; Bianchi, G; Mercuri, M; Alberghini, M; Cadossi, R.
abstract

Short intense electrical pulses transiently increase the permeability of the cell membrane, an effect known as electroporation. This can be combined with antiblastic drugs for ablation of tumours of the skin and subcutaneous tissue. The aim of this study was to test the efficacy of electroporation when applied to bone and to understand whether the presence of mineralised trabeculae would affect the capability of the electric field to porate the membrane of bone cells. Different levels of electrical field were applied to the femoral bone of rabbits. The field distribution and modelling were simulated by computer. Specimens of bone from treated and control rabbits were obtained for histology, histomorphometry and biomechanical testing. After seven days, the area of ablation had increased in line with the number of pulses and/or with the amplitude of the electrical field applied. The osteogenic activity in the ablated area had recovered by 30 days. Biomechanical testing showed structural integrity of the bone at both times. Electroporation using the appropriate combination of voltage and pulses induced ablation of bone cells without affecting the recovery of osteogenic activity. It can be an effective treatment in bone and when used in combination with drugs, an option for the treatment of metastases.

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.

2009 - ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency [Articolo su rivista]
A. V., Sauer; E., Mrak; R. J., Hernandez; E., Zacchi; Cavani, Francesco; M., Casiraghi; E., Grunebaum; C. M., Roifman; M. C., Cervi; A., Ambrosi; F., Carlucci; M. G., Roncarolo; A., Villa; A., Rubinacci; A., Aiuti
abstract

Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781. (Blood. 2009; 114: 3216-3226)

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 - Electroporation of bone tissue: Implications for use in the treatment of bone metastasis with electrochemotherapy [Articolo su rivista]
Cadossi, R.; Fini, M.; Ronchetti, M.; De Terlizzi, F.; Cavani, F.
abstract

Electrical pulses were applied to the distal femur of rabbits by inserting four electrodes in order to evaluate if and to what extent cell electroporation could be achieved in such a non-homogenous tissue. Dose response effect of number of pulses delivered was observed. 120 pulses at 1750V/cm successfully removed all osteogenetic activity among the electrodes, as evidenced by tetracyclin staining. These preclinical results lay the foundation for use of electroporation in the treatment of bone metastases. © 2009 Springer-Verlag.

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 - Cartilage repair with osteochondral autografts in sheep: Effect of biophysical stimulation with pulsed electromagnetic fields [Articolo su rivista]
F., Benazzo; M., Cadossi; Cavani, Francesco; M., Fini; G., Giavaresi; S., Setti; R., Cadossi; R., Giardino
abstract

The effect of pulsed electromagnetic fields (PEMFS) on the integration of osteochondral autografts was evaluated in sheep. After osteochondral grafts were performed, the animals were treated with PEMFs for 6 h/day or sham-treated. Six animals were sacrificed at I month. Fourteen animals were treated for 2 months and sacrificed at 6 months. At 1 month, the osteogenic activity at the transplant-host subchondral bone interface was increased in PEMF-treated animals compared to controls. Articular cartilage was healthy in controls and stimulated animals. At 6 months, complete resorption was observed in four control grafts only. Cyst-like resorption areas were more frequent within the graft of sham-treated animals versus PEMF-treated. The average volume of the cysts was not significantly different between the two groups; nevertheless, analysis of the variance of the volumes demonstrated a significant difference. The histological score showed no significant differences between controls and stimulated animals, but the percentage of surface covered by fibrous tissue was higher in the control group than in the stimulated one. Interleukin-1 and tumor necrosis factor-a concentration in the synovial fluid was significantly lower, and transforming growth factor-beta 1 was significantly higher, in PEMF-treated animals compared to controls. One month after osteochondral graft implantation, we observed larger bone formation in PEMF-treated grafts which favors early graft stabilization. In the long term, PEMF exposure limited the bone resorption in subchondral bone; furthermore, the cytokine profile in the synovial fluid was indicative of a more favorable articular environment for the graft. (c) 2008 Orthopaedic Research Society.

2008 - Effect of pulsed electromagnetic field stimulation on knee cartilage, subchondral and epyphiseal trabecular bone of aged Dunkin Hartley guinea pigs [Articolo su rivista]
Fini, M; Torricelli, P; Giavaresi, G; Aldini, N. N.; Cavani, Francesco; Setti, S; Nicolini, A; Carpi, A; Giardino, R.
abstract

It has been demonstrated that pulsed electromagnetic field (PEMF) stimulation has a chondroprotective effect on osteoarthritis (OA) progression in the knee joints of the 12-month-old guinea pigs. The aim of the present study was to discover whether the therapeutic efficacy of PEMFs was maintained in older animals also in more severe OA lesions.PEMFs were administered daily (6 h/day for 6 months) to 15-month-old guinea pigs. The knee joints (medial and lateral tibial plateaus, medial and lateral femoral condyles) were evaluated by means of a histological/histochemical Mankin modified by Carlsson grading score and histomorphometric measurements of cartilage thickness (CT), fibrillation index (FI), subchondral bone thickness (SBT) and epiphyseal bone microarchitecture (bone volume: BV/TV; trabecular thickness: Tb.Th; trabecular number: Tb.N; trabecular separation: Tb.SP). Periarticular knee bone was also evaluated with dual X-ray absorptiometry (DXA).PEMF stimulation significantly changed the progression of OA lesions in all examined knee areas. In the most affected area of the knee joint (medial tibial plateau), significant lower histochemical score (p &lt; 0.0005), FI (p &lt; 0.005), SBT (p &lt; 0.05), BV/TV (p &lt; 0.0005), Tb.Th (p &lt; 0.05) and Tb.N (p &lt; 0.05) were observed while CT (p &lt; 0.05) and Tb.Sp (p &lt; 0.0005) were significantly higher than in SHAM-treated animals. DXA confirmed the significantly higher bone density in SHAM-treated animals. Even in the presence of severe OA lesions PEMFs maintained a significant efficacy in reducing lesion progression.

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 density, elasticity, and structure on ultrasound transmission through trabecular bone cylinders [Articolo su rivista]
Cavani, Francesco; Gianluca, Giavaresi; Milena, Fini; Bertoni, Laura; Francesca de, Terlizzi; Reinhard, Barkmann; Canè, Valerio
abstract

The aim of this in vitro study is to evaluate the potentiality of quantitative ultrasound (QUS) to separate information on density, elasticity, and structure on specimens of trabecular bone. Fifteen cylinders of spongy bone extracted from equine vertebrae were progressively demineralized and subjected to QUS, micro computed tomography (mu CT), Dual energy X-ray absorptiometry (DXA) at various mineralization levels. Eventually all cylinders underwent a compression test to calculate the Young's modulus. Correlation analysis shows that speed of sound (SOS) is strictly associated to bone mineral density (BMD), Young's modulus, and all mu CT parameters except for degree of anisotropy (DA). Fast wave amplitude (FWA) is directly correlated with bone surface and total volume ratio (BS/TV) and trabecular separation (Tb Sp), and inversely correlated with trabecular number (Tb N). Because mu CT parameters were strictly correlated to BMD and Young's modulus data, partial correlation analysis was performed between SOS, FWA, and structural and elastic data in order to eliminate the effect of density. SOS was significantly correlated to bone volume and total volume ratio (BV/TV), BS/TV, and Young's modulus, and FWA was significantly correlated to Tb Sp only. These results show that SOS is strongly influenced by volumetric mineral bone density and elastic modulus of the specimen, and FWA is mainly affected by trabecular separation independently on density. Therefore, SOS and FWA are able to provide different and complementary information, at least on trabecular bone samples.

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 - Effects of electrical physical stimuli on articular cartilage [Relazione in Atti di Convegno]
Massari, L.; Benazzo, F.; Mattei, M.; Setti, S.; Fini, M.; Study Grp, Cres; Cavani, F.
abstract

Review about the physical stimulation of articular cartilage, no abstract is present. The use of physical stimuli has been the subject of several studies aimed at understanding the mechanism through which they are able to control inflammation and stimulate articular cartilage anabolic activities. Pulsed electromagnetic fields, which have been investigated for years by our group, can be easily applied to single joints without systemic effect. Their use is not indicated for the treatment of joint inflammation associated with systemic diseases such as rheumatoid arthritis. In joints, especially the knee, pulsed electromagnetic fields, unlike drugs, have the ability to completely and homogeneously permeate the whole articular cartilage and the underlying subchondral bone. Their use is aimed at controlling inflammation, stimulating the anabolic activity of the chondrocytes, and preventing cartilage degeneration, ultimately resulting in a chondroprotective activity. The treatment should lead to improvement in the overall articular function of the patient. Present clinical use of pulsed electromagnetic fields as a chondroprotector is based on preclinical and clinical research studies conducted by the CRES (Cartilage Repair and Electrical Stimulation) study group over the past seven years.

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 - Histomorphometric and mechanical analysis of the hydroxyapatite-bone interface after electromagnetic stimulation - An experimental study in rabbits [Articolo su rivista]
Fini, M; Giavaresi, G; Giardino, R; Cavani, Francesco; Cadossi, R.
abstract

We investigated the effect of stimulation with a pulsed electromagnetic field on the osseointegration of hydroxyapatite in cortical bone in rabbits. Implants were inserted into femoral cortical bone and were stimulated for six hours per day for three weeks. Electromagnetic stimulation improved osseointegration of hydroxyapatite compared with animals which did not receive this treatment in terms of direct contact with the bone, the maturity of the bone and mechanical fixation. The highest values of maximum push-out force (F-max) and ultimate shear strength (sigma(u)) were observed in the treated group and differed significantly from those of the control group at three weeks (F-max; P < 0-0001; sigma(u), p < 0.0005).

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 - Pulsed electromagnetic fields reduce knee osteoarthritic lesion progression in the aged Dunkin Hartley guinea pig [Articolo su rivista]
Fini, M; Giavaresi, G; Torricelli, P; Cavani, Francesco; Setti, S; Canè, Valerio; Giardino, R.
abstract

An experimental in vivo study was performed to test if the effect of Pulsed Electromagnetic Fields (PEMFs) on chondrocyte metabolism and adenosine A2a agonist activity could have a chondroprotective effect on the knee of Dunkin Hartley guinea-pigs of 12 months with spontaneously developed osteoarthritis (OA). After a pilot study, 10 animals were randomly divided into two groups: PEMF-treated group (6 h/day for 3 months) and Sham-treated group. Micro radiography and histomorphometry were performed on the entire articular surface of knee joints used in evaluating chondropathy severity, cartilage thickness (CT), cartilage surface Fibrillation Index (FI), subchondral bone plate thickness (SBT) and histomorphometric characteristics of trabecular epiphyseal bone. The PEMF-treated animals showed a significant reduction of chondropathy progression in all knee examined areas (p < 0.05). CT was significantly higher.(p < 0.001) in the medial tibia plateaus of the PEMF-treated group when compared to the Sham-treated group. The highest value of FI was observed in the medial tibia plateau of the Sham-treated group (p < 0.05). Significant lower values were observed in SBT of PEMF-treated group in comparison to Sham-treated group in all knee examined areas (p < 0.05). The present study results show that PEMFs preserve the morphology of articular cartilage and slower the progression of OA lesions in the knee of aged osteoarthritic guinea pigs. The chondroprotective effect of PEMFs was demonstrated not only in the medial tibial plateau but also on the entire articular surface of the knee.

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 - Behavior of the bone-titanium interface after push-in testing: A morphological study [Articolo su rivista]
Zaffe, Davide; R. Rodriguez y., Baena; S., Rizzo; C., Brusotti; M., Soncini; R., Pietrabissa; Cavani, Francesco; V., Quaglini
abstract

Fourteen titanium dental implants (Tioblast(TM)) were implanted singly in the proximal tibia of New Zealand rabbits for 120 days. A bone defect was surgically produced and filled with Bio-Oss(R) around six of these implants. After the animals were sacrificed and their organs harvested, bone segments were fixed and methacrylate embedded after the push-in test had been performed. Microradiography was performed on longitudinal sections of the implants, whereas scanning electron microscope analysis was performed on the remaining embedded half-implants using secondary electrons only. The results showed that the implants were apically and coronally surrounded by bone, whether Bio-Oss(R) was used or not. Fractures were evident through the newly formed bone and between the pre-existing and newly formed bone. Some fracture lines propagated through the bone and stopped at the implant surface without continuing along the bone-titanium interface. Detachment between the implant and the bone occurred at the coronal extremity of the implants and along its cervical region. These results highlight the fact that the bone-titanium interface has a high resistance to loading. It exhibited greater resistance than the newly formed bone and seems to behave in a manner similar to the cement lines of osteons.

2003 - Effect of trabecular orientation on mechanical resistance and ultrasound propagation in specimens of equine vertebrae [Articolo su rivista]
CAVANI, Francesco; Fini, M; De Terlizzi, F; Cadossi, M; Ciminelli, L; Ortolani, S; Cherubini, R; De Aloysio, D; Giavaresi, G; Cadossi, R; CANÈ, Valerio
abstract

Osteoporosis involves alterations, not only in density, but also in the architectural organisation of the bone; in particular, trabecular orientation, following the skeletal load directions, lends a high degree of stiffness to the whole bone. We investigated the relationship between trabecular orientation, density, stiffness and ultrasound (US) propagation in two orthogonal directions (par. = parallel to, and ort. = orthogonal to the main orientation of the trabeculae) in cylindrical equine bone specimens (thoracic vertebrae) where a preferential orientation is present. A total of 15 cylinders were progressively decalcified with 0.2 mol/L ethylenediaminetetraacetic acid (EDTA). At different levels of decalcification, we measured the apparent density (g/cm(3)), bone mineral density or BMD (g/cm(2)), stiffness coefficient (MPa) and various US parameters. Before decalcification, stiffness values were the same in all directions. As the decalcification proceeded, the stiffness declined and, at low BMD values, it was significantly different in the two directions, being the highest in the par. one. Different behaviours of US parameters were observed in the two directions: SoS (speed of sound) was closely related to apparent density, BMD and stiffness in the par. direction (r = 0.88, 0.92 and 0.88, respectively, p < 0.0001). In the ort. direction, no significant association has been found between SoS and apparent density, BMD or stiffness. In the same experimental setup, US fast wave amplitude (FWA) was related to apparent density and BMD in the par. direction (r = 0.72 and 0.67, respectively, p < 0.0001) and in the ort. direction (r = 0.83 and r = 0.84 respectively, p < 0.0001). FWA was also correlated to stiffness in both directions (r = 0.61 par.; 0.81 ort., p < 0.0001). These results show that trabecular orientation strongly influences both mechanical properties of bone and US propagation. Furthermore, we found that US parameters can be predictors of mechanical properties of the bone independent of bone density. (E-mail: fcavani@unimore.it) (C) 2003 World Federation for Ultrasound in Medicine Biology.

2002 - In vivo effects of low-frequency low energy pulsing electromagnetic fields (PEMFs) on gene expression during the inflammation phase of bone repair [Articolo su rivista]
Zucchini, P; Zaffe, Davide; Botti, P; Grande, Alexis; Cavani, Francesco; Cadossi, M; Ferrari, Sergio; Cadossi, Ruggero; Fini, M; Canè, Valerio
abstract

It has been widely demonstrated that pulsed low-frequency electromagnetic fields (PEMFs) positively affect bone repair. The aim of this study is to highlight if PEMFs influence cell metabolic activity during the replacement of the blood clot with granulation tissue in the inflammation phase of bone repair. Four equal transcortical holes were made, at the same diaphyseal level, in both metacarpals (McIII) of five mate adult horses. The left McIII were exposed to PEMFs 24 hr/day; the right untreated McIII were used as controls. Eight days after surgery, the horses were sacrificed. We investigated the effect of PEMFs on 1) histological aspects of the lesion, 2) histochemical detection of the bone marker alkaline phosphatase, and 3) molecular markers as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-beta) by reverse transcriptase-polymerase chain reaction amplification (RT-PCR). The histological analysis indicates that the blood clot, in both PEMF treated and control holes, is being replaced by granulation tissue extending from the endosteal towards the periosteal side of the lesion. TALP positive areas do not exactly correspond to the areas where fibroblasts are present, these being wider than the former. The study of the expression of the mRNA of TGF-beta1 shows no differences between treated holes and control ones. The expression of TNF-alpha and IL-6 however, is not univocal, being sometimes more expressed, sometimes less in treated or control holes. These data show that PEMFs exposure affects the expression of inflammatory cytokines (TNF-alpha and IL-6) during the very early stages of bone repair. On the contrary TFG beta expression and histological findings are not modified by PEMF exposure at least in this experimental condition.

2002 - The effect of pulsed electromagnetic fields on the osteointegration of hydroxyapatite implants in cancellous bone: a morphologic and microstructural in vivo study [Articolo su rivista]
Fini, M; Cadossi, R; Canè, Valerio; Cavani, Francesco; Giavaresi, G; Krajewski, A; Martini, L; Aldini, Nn; Ravaglioli, A; Rimondini, L; Torricelli, P; Giardino, R.
abstract

Effects of pulsed electromagnetic fields (PEMFs. 75 Hz. L6 mT) were investigated in 12 rabbits after placing hydroxyapatite (HA) implants in their femoral condyles. Six animals were stimulated with PEMFs, for three consecutive weeks, 6 h/day, while the remaining animals were sham-treated (Control Group). Rabbits were sacrificed at 3 and 6 weeks (after a 3-week non-stimulation period) For histomorphometric analysis and microhardness testing (at 200, 500, 1000, 2000 mum from the implant) around the implants. Histomorphometric analysis did not highlight any significant changes. On the contrary, there were statistically significant differences between the effects produced by PEMFs and Control Groups (r = 149.70, p = 0.0005) on the Affinity Index results as well as by the experimental time of 6 and 3 weeks (F = 17.12, p = 0.001) on the same results. In PEMF-stimulated animals the microhardness (HV) values measured in trabecular bone at a distance of 200 and 500 mum from the implants, were significantly higher with respect to controls. At 6 weeks HV values at the bone implant interface in PEMF-stimulated animals were not significantly different with respect to normal bone, while they remained significantly loader in control animals, Both morphological and structural results demonstrated a positive therapeutic effect of PEMFs in accelerating HA osteointegration in trabecular bone. (C) 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

2000 - Influence of bone tissue density and elasticity on ultrasound propagation: an in vitro study. [Articolo su rivista]
F., De Terlizzi; S., Battista; Cavani, Francesco; Canè, Valerio; R., Cadossi
abstract

Ultrasound (US) waves are mechanical vibrations that are applied to a material-bone tissue-in order to study its properties, that is, density, elasticity, and structure. In this study we evaluated in which way density and elasticity of the spongy bone influenced the transmission of 1.25 MHz US pulses. Twelve cylindrical specimens (diameter, 8 mm; height, 5 mm) excised from phalanxes of pig were decalcified with 0.5 M EDTA for different times (0, 2, and 5 days). During these periods, the samples underwent the following investigations: US transmission, density, and elasticity measurements. To assess the homogeneity of decalcification, the cross-sections of some samples were microradiographed. A detailed analysis of the US signal received was performed using velocity, Fourier analysis, and some parameters typical of signal processing technique. A good correlation was found between US velocity and density (r(2) = 0.70); a lower correlation was found between velocity and elasticity (r(2) = 0.59). If density and elasticity are considered simultaneously, the correlation with the US velocity improves significantly (r(2) = 0.84). Fourier analysis enabled us to observe a shift of the main frequency toward lower values as the decalcification process advanced. We also observed that in the regressions weighted for density, US velocity correlated poorly with elasticity (r(2) = 0.16), whereas signal processing parameters maintain a good correlation with elasticity (ultrasound peak amplitude [UPA], r(2) = 0.48; slope, r(2) = 0.62). In this study, it has been observed that when using a signal processing technique to analyze US pulses, it is possible to identify some parameters that are related in different ways to density and to elastic properties of bone. Our results show the potentiality of US technique to separate information on bone density and elasticity that X-ray-based densitometric methods do not provide.

1999 - Le fratture intracorticali del metacarpeo III nel cavallo sportivo: dimostrazione sperimentale della validità dell’osteostissi [Relazione in Atti di Convegno]
P., Botti; Cavani, Francesco; Zaffe, Davide; M., Botti; Canè, Valerio
abstract

Studio quantitativo sulla riparazione di fori transcorticali e intracorticali nel metacarpo equino.

1999 - Morphine and anandamide stimulate intracellular calcium transients in human arterial endothelial cells: coupling to nitric oxide release [Articolo su rivista]
Fimiani, C; Mattocks, D; Cavani, F; Salzet, M; Deutsch, D G; Pryor, S; Bilfinger, T V; Stefano, G B
abstract

Both morphine and anandamide significantly stimulated cultured endothelial intracellular calcium level increases in a concentration-dependent manner in cells pre-loaded with fura 2/AM. Morphine is more potent than anandamide (approximately 275 vs. 135 nM [Ca]i), and the [Ca]i for both ligands was blocked by prior exposure of the cells to their respective receptor antagonist, i.e., naloxone and SR 171416A. Various opioid peptides did not exhibit this ability, indicating a morphine-mu3-mediated process. In comparing the sequence of events concerning morphine's and anandamide's action in stimulating both [Ca]i and nitric oxide production in endothelial cells, we found that the first event precedes the second by 40+/-8 sec. The opiate and cannabinoid stimulation of [Ca]i was attenuated in cells leeched of calcium, strongly suggesting that intracellular calcium levels regulate cNOS activity.

1999 - Morphine coupling to invertebrate immunocyte nitric oxide release is dependent on intracellular calcium transients [Articolo su rivista]
Nieto-Fernandez, F E; Mattocks, D; Cavani, F; Salzet, M; Stefano, G B
abstract

Morphine significantly stimulated invertebrate immunocyte intracellular calcium level increases in a concentration-dependent manner in cells preloaded with Fura 2/AM. Morphine's action was blocked by prior exposure of the cells to the opiate receptor antagonist naloxone. Various opioid peptides did not exhibit this ability, indicating a morphine-mu 3 mediated process. In comparing the sequence of events concerning morphine's action in stimulating both [Ca2+]i and NO production in these cells, we found that the first event precedes the second by 42 +/- 7 s. The opiate stimulation of [Ca2+]i- was attenuated in cells leached of calcium. strongly suggesting that intracellular calcium levels regulate cNOS activity in invertebrate immunocytes.

1997 - Pulsed electromagnetic fields modulate enzymatic activity during the early stages of bone repair [Articolo su rivista]
Canè, Valerio; Zaffe, Davide; Cavani, Francesco; P., Botti; S., Soana
abstract

The goal of this study was to investigate whether PEMFs influence enzymatic activities during the early stages of bone repair. Two transcortical holes (4.5 mm diameter) were drilled at the same diaphyseal level in the lateral margin of the right and left metacarpal bone (McIII) of six adult male horses. The left McIII were exposed to PEMFs (75 Hz; 2.8 mT, 1.3 ms impulse width) 24 h/day; the right untreated McIII were used as controls. Horses were sacrificed 8 and 15 days after the operation. The bone segments containing the holes were fixed, dehydrated in ethanol solutions, and, undecalcified, embedded in methylmethacrylate. The midlongitudinal sections of the holes were either stained with soluidine blue or processed for evaluation of the total alkaline phosphatase (TRAP) and the tartrate-resistant acid phosphatase (TRAP). In PEMF-treated holes we found: (1) TALP is strongly positive with respect to the controls; (b) the newly formed bony trabeculae are more abundant than in the controls; (c) in both treated and control holes, no TRAP-positive osteoclasts were observed on the hole surface, whereas several osteoclasts were located on the newly formed bone tracebulae. On the basis of these data, it may be concluded that PEMFs accelerate the healing process of transcortical holes and enhance the enzymatic activity of repair tissue.

1996 - Elaboration of an experimental animal model for quantitative and qualitative studies on reparative osteogenesis [Articolo su rivista]
Cane, V.; Botti, P.; Cavani, F.; Giardino, R.; Fini, M.; Soana, S.
abstract

This paper describes an experimental animal model employed by us for evaluation of the effects of PEMFs on reparative osteogenesis. The modality and extent of reparative osteogenesis were studied in transcortical holes of equal diameter and depth, drilled at the same levels of diaphyseal and metaphyseal regions of homotypic metacarpal bones of adult horses. The macroscopic and microscopic observations show that, as usually happens in membranous ossification, all the holes, regardless of the level at which they were made, repair by deposition of woven bone tissue and along two directions: from the endosteum toward the periosteum, and concentrically to the longer axis of the hole. The quantitative data show that: (1) the amount of bone newly formed in 60 days is very similar in holes made at the same levels of homotypic bones (% = 1.5-3.7); (2) the rate of bone repair is higher in metaphyseal than in diaphyseal holes; and (3) the rate of bone deposition decreases from the endosteum toward the periosteum. Thus reparative osteogenesis proceeds with different biorhythms according to the skeletal region where it takes place, and the endosteum has a more powerful osteogenetic activity than the periosteum. The high reproducibility of the animal model described here makes it suitable for studies on electrical stimulation of reparative osteogenesis.