Alessandro Ruggeri

Publications of Alessandro Ruggeri

• Tendon and ligament fibrillar crimps give rise to left-handed helices of collagen fibrils in both planar and helical crimps.

  Authors: Marco Franchi, Vittoria Ottani, Rita Stagni, Alessandro Ruggeri

  Journal of anatomy. 03/2010; 216(3):301-9.

  Collagen fibres in tendons and ligaments run straight but in some regions they show crimps which disappear or appear more flattened during the initial elongation of tissues. Each crimp is formed ofCollagen fibres in tendons and ligaments run straight but in some regions they show crimps which disappear or appear more flattened during the initial elongation of tissues. Each crimp is formed of collagen fibrils showing knots or fibrillar crimps at the crimp top angle. The present study analyzes by polarized light microscopy, scanning electron microscopy, transmission electron microscopy the 3D morphology of fibrillar crimp in tendons and ligaments of rat demonstrating that each fibril in the fibrillar region always twists leftwards changing the plane of running and sharply bends modifying the course on a new plane. The morphology of fibrillar crimp in stretched tendons fulfills the mechanical role of the fibrillar crimp acting as a particular knot/biological hinge in absorbing tension forces during fibril strengthening and recoiling collagen fibres when stretching is removed. The left-handed path of fibrils in the fibrillar crimp region gives rise to left-handed fibril helices observed both in isolated fibrils and sections of different tendons and ligaments (flexor digitorum profundus muscle tendon, Achilles tendon, tail tendon, patellar ligament and medial collateral ligament of the knee). The left-handed path of fibrils represents a new final suprafibrillar level of the alternating handedness which was previously described only from the molecular to the microfibrillar level. When the width of the twisting angle in the fibrillar crimp is nearly 180 degrees the fibrils appear as left-handed flattened helices forming crimped collagen fibres previously described as planar crimps. When fibrils twist with different subsequent rotational angles (< 180 degrees ) they always assume a left-helical course but, running in many different nonplanar planes, they form wider helical crimped fibres.

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

  Authors: Marco Franchi, Marilisa Quaranta, Maria Macciocca, Luisa Leonardi, Vittoria Ottani, Paolo Bianchini, Alberto Diaspro, Alessandro Ruggeri

  Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA. 02/2010; 18(12):1671-8.

  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 asLigaments 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.

• Correlative microscopy of bone in implant osteointegration studies.

  Authors: Alessandra Trirè, Désirée Martini, Ester Orsini, Marco Franchi, Viviana De Pasquale, Beatrice Bacchelli, Mario Raspanti, Alessandro Ruggeri, Vittoria Ottani

  TheScientificWorldJournal. 01/2010; 10:2238-47.

  Routine morphological analyses usually include investigations by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Each of these techniquesRoutine morphological analyses usually include investigations by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Each of these techniques provides specific information on tissue morphology and all the obtained results are then combined to give an in-depth morphological overview of the examined sample. The limitations of this traditional comparative microscopy lie in the fact that each technique requires a different experimental sample, so that many specimens are necessary and the combined results come from different samples. The present study describes a technical procedure of correlative microscopy, which allows us to examine the same bone section first by LM and then, after appropriate processing, by SEM or TEM. Thanks to the possibility of analyzing the same undecalcified bone sections both by LM and SEM, the approach described in the present study allows us to make very accurate evaluations of old/new bone morphology at the bone-implant interface.

• Influence of a zirconia sandblasting treated surface on peri-implant bone healing: An experimental study in sheep.

  Authors: Beatrice Bacchelli, Gianluca Giavaresi, Marco Franchi, Désirée Martini, Viviana De Pasquale, Alessandra Trirè, Milena Fini, Roberto Giardino, Alessandro Ruggeri

  Acta biomaterialia. 02/2009;

  A sandblasting process with round zirconia (ZrO(2)) particles might be an alternative surface treatment to enhance the osseointegration of titanium dental implants. Our previous study on sheepA sandblasting process with round zirconia (ZrO(2)) particles might be an alternative surface treatment to enhance the osseointegration of titanium dental implants. Our previous study on sheep compared smooth surface titanium implants (control) with implant surfaces sandblasted with two different granulations of ZrO(2). As the sandblasted surfaces proved superior, the present study further compared the ZrO(2) surface implant with other surface treatments currently employed: machined titanium (control), titanium oxide plasma sprayed (TPS) and alumina sandblasted (Al-SL) at different times after insertion (2, 4 and 12weeks). Twelve sheep were divided into three groups of four animals each and underwent implant insertion in tibia cortical bone under general anaesthesia. The implants with surrounding tissues were subjected to histology, histomorphometry, scanning electron microscopy and microhardness tests. The experimentation indicated that at 2weeks Zr-SL implants had the highest significant bone ingrowth (p<0.05) compared to the other implant surfaces, and a microhardness of newly formed bone inside the threads significantly higher than that of Ti. The present work shows that the ZrO(2) treatment produces better results in peri-implant newly formed bone than Ti and TPS processing, whereas its performance is similar to the Al-SL surface treatment.

• Effects of freezing on the biomechanical and structural properties of human posterior tibial tendons.

  Authors: Sandro Giannini, Roberto Buda, Francesco Di Caprio, Patrizia Agati, Adriana Bigi, Viviana De Pasquale, Alessandro Ruggeri

  International orthopaedics. 05/2008; 32(2):145-51.

  This work analyzes the effects of storage by fresh-freezing at -80 degrees C on the histological, structural and biomechanical properties of the human posterior tibial tendon (PTT), used for ACLThis work analyzes the effects of storage by fresh-freezing at -80 degrees C on the histological, structural and biomechanical properties of the human posterior tibial tendon (PTT), used for ACL reconstruction. Twenty-two PTTs were harvested from eleven donors. For each donor one tendon was frozen at -80 degrees C and thawed in physiological solution at 37 degrees C, and the other was tested without freezing (control). Transmission electron microscopy (TEM), differential scanning calorimetry (DSC) and biomechanical analysis were performed. We found the following mean changes in frozen-thawed tendons compared to controls: TEM showed an increase in the mean diameter of collagen fibrils and in fibril non-occupation mean ratio, while the mean number of fibrils decreased; DSC showed a decrease in mean denaturation temperature and denaturation enthalpy. Biomechanical analysis showed a decrease in ultimate load and ultimate stress, an increase in stiffness and a decrease in ultimate strain of tendons. In conclusion fresh-freezing brings about significant changes in the biomechanical and structural properties of the human PTT. A high variability exists in the biophysical properties of tendons among individuals and in the effects of storage on tendons. Therefore, when choosing an allograft tendon, particular care is needed to choose a biomechanically suitable graft.

• Different crimp patterns in collagen fibrils relate to the subfibrillar arrangement.

  Authors: Marco Franchi, Mario Raspanti, Carlo Dell'Orbo, Marilisa Quaranta, Viviana De Pasquale, Vittoria Ottani, Alessandro Ruggeri

  Connective tissue research. 02/2008; 49(2):85-91.

  Collagen fibril ultrastructure and course were examined in different connective tissues by PLM, SEM, TEM, and AFM. In tendons, collagen fibrils were large and heterogeneous with a straightCollagen fibril ultrastructure and course were examined in different connective tissues by PLM, SEM, TEM, and AFM. In tendons, collagen fibrils were large and heterogeneous with a straight subfibrillar arrangement. They ran densely packed, parallel, and straight changing their direction only in periodic crimps where fibrils showed a local deformation (fibrillar crimps). Other tissues such as aponeurosis, fascia communis, skin, aortic wall, and tendon and nerve sheaths showed thinner uniform fibrils with a helical subfibrillar arrangement. These fibrils appeared in parallel or helical arrangement following a wavy, undulating course. Ligaments showed large fibrils as in tendon, with fibrillar crimps but less packed. Thinner uniform-sized fibrils also were observed. Fibrillar crimps seem to be related to the subfibrillar arrangement being present only in large fibrils with a straight subfibrillar arrangement. These stiffer fibrils respond mainly to unidirectional tensional forces, whereas the flexible thinner fibrils with helical subfibrils can accommodate extreme curvatures without harm, thus responding to multidirectional loadings.

• Decorin transfection induces proteomic and phenotypic modulation in breast cancer cells 8701-BC.

  Authors: Ida Pucci-Minafra, Patrizia Cancemi, Gianluca Di Cara, Luigi Minafra, Salvatore Feo, Antonella Forlino, M Enrica Tira, Ruggero Tenni, Désirée Martini, Alessandro Ruggeri, Salvatore Minafra

  Connective tissue research. 02/2008; 49(1):30-41.

  Decorin is a prototype member of the small leucine-rich proteoglycan family widely distributed in the extracellular matrices of many connective tissues, where it has been shown to play multipleDecorin is a prototype member of the small leucine-rich proteoglycan family widely distributed in the extracellular matrices of many connective tissues, where it has been shown to play multiple important roles in the matrix assembly process, as well as in some cellular activities. A major interest for decorin function concerns its role in tumorigenesis, as growth-inhibitor of different neoplastic cells, and potential antimetastatic agent. The aim of our research was to investigate wide-ranged effects of transgenic decorin on breast cancer cells. To this purpose we utilized the well-characterized 8701-BC cell line, isolated from a ductal infiltrating carcinoma of the breast, and two derived decorin-transfected clones, respectively, synthesizing full decorin proteoglycan or its protein core. The responses to the ectopic decorin production were examined by studying morphological changes, cell proliferation rates, and proteome modulation. The results revealed new important antioncogenic potentialities, likely exerted by decorin through a variety of distinct biochemical pathways. Major effects included the downregulation of several potential breast cancer biomarkers, the reduction of membrane ruffling, and the increase of cell-cell adhesiveness. These results disclose original aspects related to the reversion of malignant traits of a prototype of breast cancer cells induced by decorin. They also raise additional interest for the postulated clinical application of decorin.

• Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.

  Authors: Rossella Solmi, Mattia Lauriola, Mirko Francesconi, Désirée Martini, Manuela Voltattorni, Claudio Ceccarelli, Giampaolo Ugolini, Giancarlo Rosati, Simone Zanotti, Isacco Montroni, Gabriella Mattei, Mario Taffurelli, Donatella Santini, Furio Pezzetti, Alessandro Ruggeri, Gastone Castellani, Lia Guidotti, Domenico Coppola, Pierluigi Strippoli

  BMC cancer. 02/2008; 8:227.

  BACKGROUND: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and inBACKGROUND: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. METHODS: Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. RESULTS: Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment.In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. CONCLUSION: This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.

• Influence of different implant surfaces on peri-implant osteogenesis: histomorphometric analysis in sheep.

  Authors: Marco Franchi, Beatrice Bacchelli, Gianluca Giavaresi, Viviana De Pasquale, Désirée Martini, Milena Fini, Roberto Giardino, Alessandro Ruggeri

  Journal of periodontology. 06/2007; 78(5):879-88.

  BACKGROUND: The present study investigated peri-implant osteogenesis and implant biologic fixation in different zirconia sandblasted endosseous titanium surfaces (SLA-60 and SLA-120) and a turnedBACKGROUND: The present study investigated peri-implant osteogenesis and implant biologic fixation in different zirconia sandblasted endosseous titanium surfaces (SLA-60 and SLA-120) and a turned titanium surface (T) 2 and 4 weeks after surgery. METHODS: Seventy-two implant screws were implanted in tibia of six sheep. Histologic sections of implants (2 and 4 weeks after surgery) were analyzed with light microscopy for histomorphometric analysis of bone-to-implant contact (BIC), bone ingrowth (BI), and bone surface (BS/BV). Histologic blocks were used to perform bone microhardness studies next to the implants. Some implants were also observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: In general, the highest values of BIC, BI, BS/BV, and Vickers hardness number (HV) were measured in SLA-60 samples, followed by SLA-120 and T implants. Two weeks after surgery, all the implants appeared biologically fixed by a newly formed woven bone arranged in thin bone trabeculae and filling the gap between implant and host bone. Four weeks after implantation, the thickness of the woven bone trabeculae had increased, especially around the SLA-60 and SLA-120 implants by a gradual deposition of parallel-fiber bone. CONCLUSIONS: Our results suggest that, in the early period of peri-implant healing, the implant surface morphology that seemed to influence the increase of peri-implant osteogenesis, bone turnover, and peri-implant bone maturation was SLA-60. We suggest that this surface, characterized by moderately deep titanium cavities very similar to the osteocyte lacunae, could act as a microscopic scaffold for mesenchymal and/or osteoblast-like cells adhesion.

• Crimp morphology in relaxed and stretched rat Achilles tendon.

  Authors: Marco Franchi, Milena Fini, Marilisa Quaranta, Viviana De Pasquale, Mario Raspanti, Gianluca Giavaresi, Vittoria Ottani, Alessandro Ruggeri

  Journal of anatomy. 02/2007; 210(1):1-7.

  Fibrous extracellular matrix of tendon is considered to be an inextensible anatomical structure consisting of type I collagen fibrils arranged in parallel bundles. Under polarized light microscopyFibrous extracellular matrix of tendon is considered to be an inextensible anatomical structure consisting of type I collagen fibrils arranged in parallel bundles. Under polarized light microscopy the collagen fibre bundles appear crimped with alternating dark and light transverse bands. This study describes the ultrastructure of the collagen fibrils in crimps of both relaxed and in vivo stretched rat Achilles tendon. Under polarized light microscopy crimps of relaxed Achilles tendons appear as isosceles or scalene triangles of different size. Tendon crimps observed via SEM and TEM show the single collagen fibrils that suddenly change their direction containing knots. The fibrils appear partially squeezed in the knots, bent on the same plane like bayonets, or twisted and bent. Moreover some of them lose their D-period, revealing their microfibrillar component. These particular aspects of collagen fibrils inside each tendon crimp have been termed 'fibrillar crimps' and may fulfil the same functional role. When tendon is physiologically stretched in vivo the tendon crimps decrease in number (46.7%) (P<0.01) and appear more flattened with an increase in the crimp top angle (165 degrees in stretched tendons vs. 148 degrees in relaxed tendons, P<0.005). Under SEM and TEM, the 'fibrillar crimps' are still present, never losing their structural identity in straightened collagen fibril bundles of stretched tendons even where tendon crimps are not detectable. These data suggest that the 'fibrillar crimp' may be the true structural component of the tendon crimp acting as a shock absorber during physiological stretching of Achilles tendon.

• Self-aggregation of fibrillar collagens I and II involves lysine side chains.

  Authors: Ruggero Tenni, Myriam Sonaggere, Manuela Viola, Barbara Bartolini, M Enrica Tira, Antonio Rossi, Ester Orsini, Alessandro Ruggeri, Vittoria Ottani

  Micron (Oxford, England : 1993). 02/2006; 37(7):640-7.

  Several properties of fibrillar collagens depend on abundance and position of ionic amino acids. We recently demonstrated that N-methylation and N-acetylation of Lys/Hyl amino group did notSeveral properties of fibrillar collagens depend on abundance and position of ionic amino acids. We recently demonstrated that N-methylation and N-acetylation of Lys/Hyl amino group did not significantly alter the thermal stability of the triple helical conformation and that the binding of modified collagens I and II to decorin is lost only on N-acetylation. The positive charge at physiological pH of Lys/Hyl side chains is preserved only by N-methylation. We report here the new aspect of the influence of the same modifications on collagen self-aggregation in neutral conditions. Three collagen preparations are very differently affected by N-methylation: acid-soluble type I collagen maintains the ability to form banded fibrils with 67-nm periodicity, whereas almost no structured aggregates were detected for pepsin-soluble type I collagen; pepsin-soluble type II collagen forms a very different supramolecular species, known as segment long spacing (SLS). N-acetylation blocks the formation of banded fibrils in neutral conditions (as did all other chemical modifications reported in the literature), demonstrating that the positive charge of Lys/Hyl amino groups is essential for self-aggregation. Kinetic measurements by turbidimetry showed a sizeable increase of absorbance only for the two N-methylated samples forming specific supramolecular aggregates; however, the derivatization affects aggregation kinetics by increasing lag time and decreasing maximum slope of absorbance variation, and lowers aggregation competency. We discuss that the effects of N-methylation on self-aggregation are caused by fewer or weaker salt bridges and by decrease of hydrogen bonding potential and conclude that protonated Lys side chains are involved in the fibril formation process.

• The 3D structure of crimps in the rat Achilles tendon.

  Authors: Mario Raspanti, Alessandro Manelli, Marco Franchi, Alessandro Ruggeri

  Matrix biology : journal of the International Society for Matrix Biology. 11/2005; 24(7):503-7.

  The ultrastructure of crimps of the Achilles tendon of rat, excised and processed in a slack condition, was investigated by atomic force microscopy in air, in fluid and by scanning electronThe ultrastructure of crimps of the Achilles tendon of rat, excised and processed in a slack condition, was investigated by atomic force microscopy in air, in fluid and by scanning electron microscopy and stereo reconstruction. The tendon was made of distinct fascicles, each comprising a succession of straight segments connected by sharp angles. The length of the segments and the interposed angles varied widely. In particular, the angles ranged from almost zero to over 135 degrees . We did not observe a unique structure for the hinge regions, but rather a variety of gradations of buckling and/or torsion with no evident correlation with other features of tendon. A constant hallmark was the local loss of regular molecular packing, as revealed by the disappearance of the D-banding. Our results do not support recent reports of a helical structure or smooth sinusoidal waves in tendons. Such structures may nonetheless exist in other non-tensile structures whose collagen fibrils exhibit a helical inner architecture and are able to follow a highly convoluted course without buckling or crimping.

• Different titanium surface treatment influences human mandibular osteoblast response.

  Authors: Stefano Guizzardi, Carlo Galli, Desiree Martini, Silvana Belletti, Anna Tinti, Mario Raspanti, Paola Taddei, Alessandro Ruggeri, Renato Scandroglio

  Journal of periodontology. 03/2004; 75(2):273-82.

  BACKGROUND: Six titanium disks with six different surface treatments were examined: SS: smooth (polished) surface; TPS: plasma spray; C100: sand blasting by aluminum oxide (Al2O3) diameter 100 micromBACKGROUND: Six titanium disks with six different surface treatments were examined: SS: smooth (polished) surface; TPS: plasma spray; C100: sand blasting by aluminum oxide (Al2O3) diameter 100 microm and acid etching; C150: sand blasting by Al2O3 diameter 150 microm and acid etching; B60: sand blasting by zirconium oxide (ZrO2) diameter 60 microm and acid etching; and B120: sand blasting by ZrO2 diameter 120 microm and acid etching. METHODS: The surface characteristics were determined by scanning electron microscopy (SEM) observation and a roughness tester. Raman spectroscopy was used to determine the presence of residual substances on the samples. Cells were seeded onto the disk and after 24 hours, 6 days, and 12 days were observed under SEM and growth curves generated with a cell counter. Some samples were used to determine alkaline phosphatase activity (ALP), using a colorimetric assay. RESULTS: SEM observation revealed drastic differences in surface microtopography, with a higher cell density on sand-blasted and acid-etched (SLA) samples than SS and TPS, and more regularly aligned cells on B60 and B120 surfaces than on the others. The growth curves showed a greater adhesion of cells on the etched/blasted surfaces compared to the SS and TPS surfaces. The number of cells increased on all the SLA samples, especially B60, throughout the experiment. At the same time, there was considerable ALP activity on the B60 sample, while it remained at extremely low levels on SS and TPS surfaces. Raman analyses revealed Al2O3 debris on C100 and C150, partly explaining the poorer performances of these two surface treatments, since this substance was shown to be toxic for cultured osteoblasts. CONCLUSIONS: Surface treatments influence the growth and the metabolic activity of cultured osteoblasts, and B60 seems to be the most favorable surface inducing a more pronounced proliferation of cells together with a high differentiation degree.

• Osteogenesis and morphology of the peri-implant bone facing dental implants.

  Authors: Marco Franchi, Ester Orsini, Alessandra Trire, Marilisa Quaranta, Desiree Martini, Gabriella G Piccari, Alessandro Ruggeri, Vittoria Ottani

  TheScientificWorldJournal. 02/2004; 4:1083-95.

  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 healingThis 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.

• Collagen fibril surface structures: freeze-etching data and computer modelling

  Authors: Mario Raspanti, Fulvia Ortolani, Alessandro Ruggeri, Maurizio Marchini

  International Journal of Biological Macromolecules.

  In order to investigate the correlation between primary structure and surface profile of collagen, freeze-fractured and deep-etched (dehydrated) collagen fibrils have been compared with a fibrilIn order to investigate the correlation between primary structure and surface profile of collagen, freeze-fractured and deep-etched (dehydrated) collagen fibrils have been compared with a fibril model built directly from the amino acid sequence. The model appeared to be almost identical to the freeze-etched collagen fibril with respect to the most relevant features, such as the gap-overlap ratio and the location of the two main intraperiod ridges (X3 and X2). However, it did not show the minor ridges observed in the gap zone (X1 and Y1), thus suggesting that these structures, although consistently present in collagen fibrils, may in fact be due to post-translational modifications or to non-collagenic material adhering to the fibril.

• Direct Visualization of Collagen-Bound Proteoglycans by Tapping-Mode Atomic Force Microscopy

  Authors: Mario Raspanti, Andrea Alessandrini, Vittoria Ottani, Alessandro Ruggeri

  Journal of Structural Biology.

  Most studies on the interaction of collagen with proteoglycans, two universal components of connective tissues, use technical approaches which substantially modify the shape and size of theMost studies on the interaction of collagen with proteoglycans, two universal components of connective tissues, use technical approaches which substantially modify the shape and size of the proteoglycans themselves. In the present study unfixed, untreated collagen fibrils from rat tail tendon were dehydrated and observed by tapping-mode atomic force microscopy. The surface of collagen fibrils immediately reveals a periodic alternation of gap and overlap zones. A thin, transverse ridge decorates the gap zone, while other filamentous structures run on the fibril surface, either parallel or perpendicular to the fibril axis. These surface structures are much enhanced by Cupromeronic Blue preincubation, while pretreatment with chondroitinase ABC removes them completely, leaving barely detectable transverse ridges. The ridge and filaments are likely to represent, respectively, the core protein and the glycosaminoglycan side chains of proteoglycans, displayed with a far better resolution than with conventional histochemical or immunohistochemical techniques. Our data suggest that proteoglycan molecules are capable of different, multiple interactions with the collagen fibril surface as well as with each other.

• Low-temperature heat-deproteinated compact bone to heal large bone defects

  Authors: Stefano Guizzardi, Mario Raspanti, Désirèe Martini, Renato Scandroglio, Paolo Govoni, Alessandro Ruggeri

  Biomaterials.

  The potential of low-temperature (400 °C), heat-treated bone matrix in osteorepair has been evaluated in vivo by implantation into defects artificially created in rodent tibia. Histological andThe potential of low-temperature (400 °C), heat-treated bone matrix in osteorepair has been evaluated in vivo by implantation into defects artificially created in rodent tibia. Histological and ultrastructural analysis of the bone-implant interface has been carried out on samples obtained at 1 to 6 weeks from operation. The obtained data showed that calcined bone is well tolerated and does not cause acute or chronic inflammatory reactions. Osteoid tissue, tightly adhered to the implant, appears within 2 weeks of the operation, while after 6 weeks newly formed bone surrounds and infiltrates the implant. Of greater note, the detection of good adhesion between bone and implant ultrastructurally is demonstrated by the absence of fibrillar connective tissue at the interface. For these reasons, our preliminary observations suggest that low-temperature calcined bone (biological apatite or heatdeproteinated bone) may have a rightful place among the osteointegrators.

• Different architectures of the collagen fibril: morphological aspects and functional implications

  Authors: Mario Raspanti, Vittoria Ottani, Alessandro Ruggeri

  International Journal of Biological Macromolecules.

  Several tissues known to contain collagen fibrils with a ‘helical’ arrangement were studied by t.e.m. and freeze-fracture. In all the tissues examined, the diameter of the collagen fibrils appearedSeveral tissues known to contain collagen fibrils with a ‘helical’ arrangement were studied by t.e.m. and freeze-fracture. In all the tissues examined, the diameter of the collagen fibrils appeared to be tissue-specific and fairly constant within the same tissue. No statistical differences, on the contrary, were detectable in the coiling angle which appeared similar in all the tissues and independent of both diameter and age of the fibril. Rat tail tendon was also examined under the same technical conditions and showed collagen fibrils of large and very heterogeneous diameter and with a consistent ‘straight’ arrangement. These data seem to suggest that the ‘helical’ and ‘straight’ arrangements may actually identify different types of collagen fibrils. The authors discuss the possible functional significance of these arrangements and present two hypotheses on the three-dimensional structure of the ‘helical’ fibril.

• Structure relates to elastic recoil and functional role in quadriceps tendon and patellar ligament

  Authors: Marco Franchi, Marilisa Quaranta, Maria Macciocca, Viviana De Pasquale, Vittoria Ottani, Alessandro Ruggeri

  Micron.

  Tendons and ligaments have similar but slightly different structure and composition. Crimps of tendons and ligaments are morphological structures related to the elastic functional properties of theseTendons and ligaments have similar but slightly different structure and composition. Crimps of tendons and ligaments are morphological structures related to the elastic functional properties of these connective tissues. Aim of this study was to investigate the morphological arrangement of collagen fibres, fibrils and crimping pattern of suprapatellar (rectus femoris tendon-RFT and vastus intermedius tendon-VIT) and infrapatellar connective tissues (patellar ligament-PL) to relate their structural aspects to their common function role of leg extension. RFT, VIT and PL were removed from knees of Sprague–Dawley rats and light and electron microscopy (TEM and SEM) performed. Sagittal sections showed that collagen array and crimping pattern were similar in RFT and PL but differed from VIT. Morphometric analysis confirmed that crimp number was about the same in RFT and PL (5.4 ± 1.4 and 6.1 ± 2.8 respectively), but it was almost three times higher in VIT (14.5 ± 4.7). Similarly crimp top angle in RFT and PL (141.5 ± 15.0° and 146.2 ± 12.2° respectively) was significantly higher than in VIT (122.3 ± 14.8°) and the crimp base length was more than twice as wide in RFT (75.5 ± 22.6 μm) and PL (72.3 ± 28.9 μm) than in VIT (36 ± 14.1 μm). The smaller, fewer and most crimped crimps in VIT show that this tendon has a greater elastic recoil and responds to higher forces as among quadriceps muscles the vastus intermedius belly contributes the most during knee extension. By contrast, RFT acting as a “stopper” tendon also plays a ligament role by limiting an excessive flexion of the joint during postural rest position of the knee.