Alessandro Ruggeri

Hierarchical structures in fibrillar collagens

The collagen family includes several large transcripts, usually exceeding 1000 amino acid residues per single chain. As a group, they make up 1/3 of all the protein of the body and are responsible for modelling the framework of connective tissues; individually, they show both a wide variety and a complex hierarchy of mutual interactions, and form a range of functional aggregates including a variety of fibrils, microfibrils and basal membranes. Of the collagens, the fibril-forming types (i.e. the types I, II III, V and XI) are the most abundant and the most extensively studied. At the primary structure level, the amino acid sequence of all collagens is now known in detail and it shows a distinctive domain organization, its composition being dominated by the amino acid glycine (roughly 1/3 of all residues) and by post-translational hydroxylation of proline and lysine residues. Collagen secondary and tertiary structure, which together give origin to a classic triple helix, were painstakingly determined in the 1950s and 1960s. In contrast with the primary, secondary and tertiary structure, the supramolecular arrangement within collagen fibres seems to be far more elusive, and none of the models so far advanced can be said to be universally accepted. Half a century of research and debate spawned numerous mutually incompatible models, most of them focussing either on a quasi-crystalline supramolecular array or on several forms of microfibrillar aggregates, while radial fibrils, epitaxial fibrils and other structural models have almost been ignored. In many cases, data gained with a single technique from a single tissue were arbitrarily given a general legitimacy, whilst other well-documented morphological evidence went virtually unnoticed by the scientific community.Moreover, in recent years there has been a growing interest in the multiple interactions of collagens with the other macromolecules of the extra-cellular matrix, as their structure and their functional role become known. It is now indisputable that collagen interacts and forms functional entities with several other macromolecules of the extracellual matrix. This paper will succinctly review some current concepts on the structural biology of collagen higher-order structures.

Crimp morphology in relaxed and stretched rat Achilles tendon.

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 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° in stretched tendons vs. 148° 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.

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

This work analyzes the effects of storage by fresh-freezing at −80°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°C and thawed in physiological solution at 37°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.RésuméCe travail a pour but d’analyser les effets du stockage à −80°C sur le plan histologique, structurel et biomécanique d’un tendon le tibial ou jambier postérieur (PTT), utilisé pour la reconstruction des ligaments croisés antérieurs. 22 PTT ont été conservées provenant de 11 donneurs. Pour chaque donneur un tendon a été congelé à −80°C et l’autre, conservé dans une solution physiologique à 37°C. Ces tendons ont été testés. L’examen par microscope électronique (TEM), le scanner calorimétrique (DSC) et une analyse biomécanique ont été réalisés. Nous avons trouvé des changements dans les tendons conservés au froid en comparaison du groupe contrôle. Le TEM, examen au microscope électronique a montré une diminution du diamètre des fibres collagènes. L’analyse biomécanique a montré également une diminution de la résistance à la charge et au stress ainsi qu’une augmentation de la rigidité et une diminution des contraintes terminales au niveau du tendon. En conclusion: la congélation des tendons frais amène des modifications significatives des caractéristiques biomécaniques et structurelles du tendon PTT humain. Il existe une variation importante des propriétés biophysiques des tendons parmi les individus et du fait de leurs conservations. Pour cela, il est nécessaire lorsque l’on choisit un tendon et une allogreffe du tendon d’apporter dans le choix sur le plan biomécanique un soin particulier.

Implants of heterologous demineralized bone matrix for induction of posterior spinal fusion in rats

The authors tested The osteoinductive capacity of powdered heterologous (bovine) demineralized bone matrix in rats. The first part of the study concerned a monolateral posterior spinal implant after decortication of three vertebrae, using as a control area the animals contralateral side, in which neither bone graft nor any other material were placed, In another group of rats, a comparative evaluation was made of powdered heterologous demineralized bone matrix and fresh autologous bone. In the same animal, autologous bone was implanted to realize a thoracic posterior fusion and demineralized bone matrix, to induce a posterior fusion in the lumbar area. All data obtained suggested a good osteoinductive activity of heterologous powdered demineralized bone matrix. The two posterior spinal fusions done in the same animal with heterologous demineralized bone matrix or authologous bone, respectively, had similar callus development and required the same fine for formation.

Collagen fibrils with straight and helicoidal microfibrils: a freeze-fracture and thin-section study.

Replicas of freeze-fractured collagen fibrils of rat aorta, dermis, perichondrium (of rib and tracheal cartilage), and peritendineum (of the tail tendon) and of pig cornea show microfibrils which are helicoidally arranged; however, microfbrils have a straight arrangement in the cartilage of rat rib and trachea as well as in that of bovine knee joint and nasal septum. The microfibrils of rat tail tendon collagen also follow a straight course. In thin sections, after the specimens were treated with a 4 M guanidinium chloride solution, the helicoidal arrangement of the microfibrils in the collagen of the aorta and dermis and their straight course in the collagen of the nasal septum cartilage and tail tendon are also demonstrated.

Biomechanical and histomorphometric investigations on two morphologically differing titanium surfaces with and without fluorohydroxyapatite coating: an experimental study in sheep tibiae

The influence of fluorohydroxyapatite (FHA) coating and surface roughness of Ti6Al4V implants on bone response was investigated. Uncoated and FHA-coated screws with lower (LR and LR+FHA; Ra: 5.7+/-0.2 microm) and higher (HR and HR+FHA; Ra: 21.8+/-0.9 microm) surface roughness, were inserted into the diaphyses of 8 sheep tibiae. Twelve weeks after implantation, extraction torque and bone-to-implant contact were evaluated. The smoothest surfaces showed an improved extraction torque and significant differences were observed between LR and HR (-24.6%, p<0.0005), LR and HR+FHA (-30.7%, p<0.0005), LR+FHA and HR (-17.4%, p<0.005), and LR+FHA and HR+FHA (-24.0%, p<0.005). The bone-to-implant contact data paralleled the biomechanical data: the smoother the surface, the greater the bone-to-implant contact. Significant (p<0.0005) decreases in bone-to-implant contact were observed between LR+FHA and HR (-24.2%), and between LR+FHA and HR+FHA (-29.2%). The current findings suggest that LR surfaces significantly improve the osteointegration rate of implanted cortical screws independently of the FHA coating.

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

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 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° 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°) they always assume a left‐helical course but, running in many different nonplanar planes, they form wider helical crimped fibres.

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

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 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.

Collagen fibril surface : TMAFM, FEG-SEM and freeze-etching observations

Native, unfixed collagen fibrils from rat tail tendon were dehydrated following different procedures and observed under a FEG‐SEM and an AFM operated in Tapping Mode (TMAFM). Freeze‐etched, untreated fibrils from the same tissue were also observed for comparison. The most notable features of the fibril surface, i.e., the gap/overlap alternation and three prominent intraperiod ridges, were simultaneously visible only in freeze‐etched specimens, while under the SEM and the TMAFM their appearance was dependent on both the dehydration procedure and the visualization technique. The different susceptibility of the collagen fibril surface structures to various treatments clearly implies the existence of domains of different composition. Moreover, identical specimens were imaged differently by SEM and TMAFM, highlighting instrument‐specific advantages and limitations. The onset of dehydration‐dependent, procedure‐specific artifacts should be considered in high‐resolution studies of connective tissues. As for any biological specimen, the final aspect of collagen fibrils is determined no less by the preliminary treatments than by the visualization approach.

Different Crimp Patterns in Collagen Fibrils Relate to the Subfibrillar Arrangement

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 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.