Marilisa Quaranta

Osteogenesis and Morphology of the Peri-Implant Bone Facing Dental Implants

This study investigated the influence of different implant surfaces on peri-implant osteogenesis and implant face morphology of peri-implant tissues during the early (2 weeks) and complete healing period (3 months). Thirty endosseous titanium implants (conic screws) with differently treated surfaces (smooth titanium = SS, titanium plasma sprayed = TPS, sand-blasted zirconium oxide = Zr-SLA) were implanted in femur and tibiae diaphyses of two mongrel sheep. Histological sections of the implants and surrounding tissues obtained by sawing and grinding techniques were observed under light microscopy (LM). The peri-implant tissues of other samples were mechanically detached from the corresponding implants to be processed for SEM observation. Two weeks after implantation, we observed osteogenesis (new bone trabeculae) around all implant surfaces only where a gap was present at the host bone-metal interface. No evident bone deposition was detectable where threads of the screws were in direct contact with the compact host bone. Distance osteogenesis predominated in SS implants, while around rough surfaces (TPS and Zr-SLA), both distance and contact osteogenesis were present. At SEM analysis 2 weeks after implantation, the implant face of SS peri-implant tissue showed few, thin, newly formed, bone trabeculae immersed in large, loose, marrow tissue with blood vessels. Around the TPS screws, the implant face of the peri-implant tissue was rather irregular because of the rougher metal surface. Zr-SLA screws showed more numerous, newly formed bone trabeculae crossing marrow spaces and also needle-like crystals in bone nodules indicating an active mineralising process. After 3 months, all the screws appeared osseointegrated, being almost completely covered by a compact, mature, newly formed bone. However, some marrow spaces rich in blood vessels and undifferentiated cells were in contact with the metal surface. By SEM analysis, the implant face of the peri-implant tissue showed different results. Around the SS screws, the compact bone with areas of different mineralisation rate appeared very smooth, while around the rougher TPS screws, the bone still showed an irregular surface corresponding to the implant macro/microroughness. Around the Zr-SLA screws, a more regular implant-bone surface and sparse, calcified marrow spaces were detectable.Results from this research suggest that 2 weeks after implantation, trabecular bone represents the calcified healing tissue, which supports the early biological fixation of the implants. The peri-implant marrow spaces, rich in undifferentiated cells and blood vasculature, observed both 2 weeks and 3 months after surgery, favour the biological turnover of both early and mature peri-implant bone. The implant surface morphology strongly influences the rate and the modality of peri-implant osteogenesis, as do the morphology and arrangement of the implant face in peri-implant bone both during early healing (after 2 weeks) and when the implant is just osseointegrated; rough surfaces, and in particular Zr-SLA, seem to better favour bone deposition on the metal surface.

An increased expression of PI-PLCβ1 is associated with myeloid differentiation and a longer response to azacitidine in myelodysplastic syndromes

This study tested the hypothesis that PI‐PLCβ1 is associated with myeloid differentiation and that its expression could be useful for predicting the response of MDS patients to azacitidine, as the clinical effect of epigenetic treatments is often detectable only after several cycles of therapy. To this end, PI‐PLCβ1 was quantified on 70 MDS patients (IPSS risk: 13 Low, 20 Int‐1, 31 Int‐2, 6 High) at baseline and during the first 3 cycles of azacitidine. Results were then compared with the hematologic response, as assessed after the sixth cycle of azacitidine therapy. Overall, 60 patients completed 6 cycles of azacitidine, and for them, a clinical and molecular evaluation was possible: 37 of these patients (62%) showed a specific increase of PI‐PLCβ1 mRNA within the first 3 cycles, which was associated with a longer duration of response and with an increased myeloid differentiation, as evidenced by PI‐PLCγ2 induction and the recruitment of specific myeloid‐associated transcription factors to the PI‐PLCβ1 promoter during azacitidine response. Moreover, the increase of cyclin D3 gene expression throughout all of the therapy showed that PI‐PLCβ1‐dependent signaling is indeed activated in azacitidine responder patients. Taken together, our results show that PI‐PLCβ1 quantification in MDS predicts the response to azacitidine and is associated with an increased myeloid differentiation.

Collagen fibre arrangement and functional crimping pattern of the medial collateral ligament in the rat knee

Ligaments have been described as multifascicular structures with collagen fibres cross-connecting to each other or running straight and parallel also showing a waviness or crimping pattern playing as a shock absorber/recoiling system during joint motions. A particular collagen array and crimping pattern in different ligaments may reflect different biomechanical roles and properties. The aim of the study was to relate the 3D collagen arrangement in the crimping pattern of the medial collateral ligament (MCL) to its functional role. The MCL is one of the most injured ligaments during sports activities and an experimental model to understand the rate, quality and composition of ligaments healing. A deep knowledge of structure–function relationship of collagen fibres array will improve the development of rehabilitation protocols and more appropriate exercises for recovery of functional activity. The rat MCL was analysed by polarized light microscopy, confocal laser microscopy and scanning electron microscopy (SEM). Histomorphometric analysis demonstrated that MCL crimps have a smaller base length versus other tendons. SEM observations demonstrated that collagen fibres showing few crimps were composed of fibrils intertwining and crossing one another in the outer region. Confocal laser analyses excluded a helical array of collagen fibres. By contrast, in the core portion, densely packed straight collagen fibres ran parallel to the main axis of the ligament being interrupted both by planar crimps, similar to tendon crimps, and by newly described right-handed twisted crimps. It is concluded that planar crimps could oppose or respond exclusively to tensional forces parallel to the main ligament axis, whereas the right-handed twisted crimps could better resist/respond to a complex of tensional/rotational forces within the ligament thus opposing to an external rotation of tibia.

Strategic Role of Nuclear Inositide Signalling in Myelodysplastic Syndromes Therapy

Nuclear inositide signalling is implicated in normal and pathological cell proliferation and differentiation in several distinct models. Among the key molecules of nuclear inositide pathways, phosphoinositide-phospholipase (PI-PLC) C β1 is essential for regulating hematopoiesis, particularly along myeloid and erythroid lineage. Moreover, Akt activation is associated with protein synthesis, via mTOR pathway, and with erythroid induction, through PI-PLCγ1 activation. Myelodysplastic syndromes (MDS) are a series of heterogeneous diseases characterized by ineffective hemopoiesis, with a variable risk of evolution into acute myeloid leukemia (AML). Therapeutic approaches for MDS include demethylating agents, such as azacitidine, aiming at reducing cell proliferation, and erythropoietin, useful for sustaining a normal erythropoiesis. In the last few years, a role for nuclear inositide signalling as a therapeutic target in MDS has been disclosed, in that PI-PLCβ1 increase is associated with azacitidine responsiveness, even when this drug is used in combination with other agents, and Akt is specifically activated in MDS at higher risk of AML evolution. On the other hand, recent data demonstrated that inositide signalling can also be involved in erythroid therapy, given the inhibitory effect of erythropoietin on PI-PLCβ1 and the activation of Akt/PI-PLCγ1 pathway, following the administration of erythropoietin. Here, we review the strategic role of nuclear inositide signalling in MDS, in pathogenesis and therapy.

Contribution of glycosaminoglycans to the microstructural integrity of fibrillar and fiber crimps in tendons and ligaments.

The biomechanical roles of both tendons and ligaments are fulfilled by the extracellular matrix of these tissues. In particular, tension is mainly transmitted and resisted by protein (collagen, elastin) fibers, whereas compression is opposed by water-soluble glycosaminoglycans (GAGs). GAGs spanning the interfibrillar spaces and interacting with fibrils through the interfibrillar proteoglycans also seem to play a part in transmitting and resisting tensile stresses. Both tendons and ligaments showing similar composition, but different functional roles and collagen array, exhibit periodic undulations of collagen fibers or crimps. Each crimp is composed of many knots of each single fibril or fibrillar crimps. Fibrillar and fiber crimps play a mechanical role in absorbing the initial loading during elongation of both tendons and ligaments, and in recoiling fibrils and fibers when tissues have to return to their original length. This study investigated whether GAGs covalently attached to proteoglycan core proteins directly affect the 3D microstructural integrity of fibrillar crimp regions and fiber crimps in both tendons and ligaments. Achilles tendons and medial collateral ligaments of the knee from eight female Sprague-Dawley rats (90 days old) incubated in a chondroitinase ABC solution to remove GAGs were observed under a scanning electron microscope (SEM). In addition, isolated fibrils of these tissues obtained by mechanical disruption were analyzed by a transmission electron microscope (TEM). Both Achilles tendons and medial collateral ligaments of the rats after chemical or mechanical removal of GAGs still showed crimps and fibrillar crimps comparable to tissues with a normal GAG content. All fibrils in the fibrillar crimp region always twisted leftwards, thus changing their running plane, and then sharply bent, changing their course on a new plane. These data suggest that GAGs do not affect structural integrity or fibrillar crimp functions that seem mainly related to the local fibril leftward twisting and the alternating handedness of collagen from a molecular to a supramolecular level.

Early healing events around titanium implant devices with different surface microtopography: a pilot study in an in vivo rabbit model.

In the present pilot study, the authors morphologically investigated sandblasted, acid-etched surfaces (SLA) at very early experimental times. The tested devices were titanium plate-like implants with flattened wide lateral sides and jagged narrow sides. Because of these implant shape and placement site, the device gained a firm mechanical stability but the largest portion of the implant surface lacked direct contact with host bone and faced a wide peri-implant space rich in marrow tissue, intentionally created in order to study the interfacial interaction between metal surface and biological microenvironment. The insertion of titanium devices into the proximal tibia elicited a sequence of healing events. Newly formed bone proceeded through an early distance osteogenesis, common to both surfaces, and a delayed contact osteogenesis which seemed to follow different patterns at the two surfaces. In fact, SLA devices showed a more osteoconductive behavior retaining a less dense blood clot, which might be earlier and more easily replaced, and leading to a surface-conditioning layer which promotes osteogenic cell differentiation and appositional new bone deposition at the titanium surface. This model system is expected to provide a starting point for further investigations which clarify the early cellular and biomolecular events occurring at the metal surface.

Multi-layer spiral CT with 2D, 3D and 4D volume rendered electronic reconstructions of wax models and natural bone made by Giuseppe Astorri kept at “Luigi Cattaneo” Museum in Bologna

The Museum’s collection of normal and pathological wax anatomical models provides a clear understanding of the developments in medical knowledge that took place during the 18th and 19th centuries. In this period the interest of the anatomists began to move from normal anatomy to pathological anatomy. The wax modelers made both types of wax anatomical models: normal and pathological. Our study investigates through the works of Giuseppe Astorri the differences between these two types of models, revealing hidden structures and materials used in a completely non-invasive way. The Computer Tomography (CT) analysis was carried out using an experimental CT system specifically designed for the analysis of Cultural Heritage materials, developed by the X-ray imaging research group at the Physics and Astronomy Department of the University of Bologna. The results of this project can also be shown through a dynamic 3D (i.e. 4D) virtual projection using a device capable of emulating a holographic representation.

Investigation of human cadavers: one year of anatomic variants and their clinical correlation

During the last year we found a plethora of variants while performing anatomical dissection on human cadavers. In the present report we describe the previously quoted variation morphologies focusing on their clinical relevance. The first variant observed was a left vertebral artery (VA) arising from the aortic arch, it is present in the 5% to the 8 % of the individuals. The left VA of aortic origin showed a remarkably higher incidence of arterial dissection than left VA of a left subclavian artery origin. Also, the present pattern has to be taken into consideration before any intervention in the local region as to avoid unexpected events in relation to the aberrant vertebral artery. A second dissection study led to the finding of the right posterior communicating artery of the Circle of Willis absence. This morphology may be a relevant risk factor for ischemic cerebral infarction if the patient suffers of internal carotid artery occlusion or severe stenosis. The same cadaver presented another variant: anterior inferior cerebellar artery bilateral absence. While dissecting the vein of abdomen and thorax, we discover other two variants: bilateral iliolumbar veins draining into the testicular veins and three pulmonary veins entering into the right side of the left atrium. Cognition of the first variation it is critical during the anterior approach for spinal procedures, it will help surgeons to anticipate and to avoid potentially catastrophic complication such massive haemorrhages due to an avulsion of an unexpected extra vein; whether the knowledge of the pulmonary veins variant, compatible with R3a Marom’s classification pattern, is essential for guidance during paroxysmal atrial fibrillation ablation procedures and preclude perioperative bleeding in video-assisted thoracoscopic surgery (VATS). Anatomical variant findings enlarge the current knowledge of anatomists, but also the cognition of surgeons and physicians that need to be aware and always up to date on the incidence level of the variable course of arteries, veins, and nerves especially while performing clinical tests and surgical operations.

Hypo and retrotympanum: the importance of anatomical variants

The hypo- and retrotympanum host a variety of crucial anatomical structures1, characterized by high variability, which are poorly been described. The aim of our study is to describe and classify the anatomical variants of the hypo- and retrotympanum by the means of transcanal endoscopy2. We hypothesize that the retro- and hypotympanum are subject to more anatomical variability than actually thought. Moreover, the configuration as bridge variants and variably shaped sinus interconnects the different subregions. A total of 125 middle ears (83 cadaveric dissections) were explored by the means of 3mm straight and angled scopes. The variants were documented photographically and tabularized. The bony crests ponticulus, subiculum and finiculus1 were most frequently represented as ridges. The ponticulus showed the highest variability with 38% ridge, 35% bridge and 27% incomplete presentation. The subiculum was bridge - shaped only in 8% of the cases, while the finiculus in 17%. The sinus tympani had a normal shape in 66% of the cases. A subcochlear canaliculus was observed in 50%. The retro- and hypotympanum were classified respectively to the present bony crests and sinus in chambers type I to IV. In our opinion, the retro- and hypotympanum have to be considered as a tightly coherent region of the middle ear. For this purpose, we propose a straightforward classification, according to the presence of the different bony crests and sinus forming the different chambers of the retro- and hypotympanum. The introduced classification may also serve as intraoperative assessment, to be aware of the different anatomical subregions. The hidden areas of the retro- and hypotampanum are difficult to access and therefore represent a region of risk for residual cholesteatomatous disease after surgical treatment. The extension below a bridge bony crest or into a deep sinus demands thorough exploration; therefore, exact anatomical knowledge and an effective technique to visualize the whole middle ear are required.

Differential activation of nuclear inositide-dependent signalling pathways during erythropoiesis and myelopoiesis induced by lenalidomide and azacitidine in myelodysplastic syndromes (MDS)

Inositide-dependent signalling pathways regulated by phosphoinositide-specific phospholi- pase C (PI-PLC) beta1 have been demonstrated to play important roles in MDS pathogenesis and in cell differentiation (1). Moreover, the MDS therapy aims at inducing myeloid and/or erythroid differentiation of MDS stem cells. Indeed, azacitidine is a demethylating agent that can induce myeloid differentiation. On the other hand, lenalidomide may restore a normal erythropoiesis. The exact molecular mechanisms underlying the effect of azacitidine and lenalidomide in MDS cells are still unclear, although it is clear that these therapies regulate stem cell proliferation, differentiation and apoptosis (2). The combination of azacitidine and lenalidomide in MDS therapy is now under considera- tion, given the capability of both drugs to balance proliferation and differentiation processes (3). In this study we analyzed the molecular effect of this combination therapy on PI-PLC isoenzymes, not only studying PI-PLCbeta1, but also PI-PLCgamma1, that can be associated with erythropoiesis. We analyzed 44 patients diagnosed with high-risk MDS who were given azacitidine and lenalidomide. Given the limited number of cells, we quantified the expression of these molecules by Real-Time PCR analyses and immunocytochemical experiments. Moreover, we carried out cell cycle analyses and studied both PI-PLCbeta1 methylation status and the expression of Globin genes. In our case series, 28/44 patients were evaluable, with an overall response rate of 78.6% (22/28 cases). At a molecular level, a significant increase of PI-PLCbeta1 and/or PI-PLCgamma1 expression was associated with a favourable clinical response to the combination therapy. Responder cases also showed an increase of Beta-globin expression, hinting at a specific contri- bution of lenalidomide on erythroid activation, whilst the frequent demethylation of PI-PLCbeta1 promoter could be specifically linked to azacitidine. Taken together, our results show that the combination of azacitidine and lenalidomide can be important for activating PI-PLC isoenzymes, therefore regulating myeloid and erythroid dif- ferentiation in MDS cells.