Vittoria Ottani

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.

Detachment of titanium and fluorohydroxyapatite particles in unloaded endosseous implants

The shape, surface composition and morphology of orthopaedic and endosseous dental titanium implants are key factors to achieve post-surgical and long-term mechanical stability and enhance implant osteointegration. In this study a comparison was made between 12 titanium screws, plasma-spray-coated with titanium powders (TPS), and 12 screws with an additional coating of fluorohydroxyapatite (FHA-Ti). Screws were implanted in the femoral and tibial diaphyses of two mongrel sheep and removed with peri-implant tissues 12 weeks after surgery. The vibrational spectroscopic, ultrastructural and morphological analyses showed good osteointegration for both types of implants in host cortical bone. The portion of the FHA-Ti implants in contact with the medullary canal showed a wider area of newly formed peri-implant bone than that of the TPS implants. Morphological and EDAX analyses demonstrated the presence of small titanium debris in the bone medullary spaces near the TPS surface, presumably due to the friction between the host bone and the implant during insertion. Few traces of titanium were detected around FHA-Ti implants, even if smaller FHA debris were present. The present findings suggest that the FHA coating may act as a barrier against the detachment of titanium debris stored in the medullary spaces near the implant surface.

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.

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.

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.

Collagen fibril arrangement and size distribution in monkey oral mucosa

Collagen fibre organisation and fibril size were studied in the buccal gingival and hard palate mucosa of Macacus rhesus monkey. Light and electron microscopy analysis showed connective papillae exhibiting a similar inner structure in the different areas examined, but varying in distribution, shape and size. Moving from the deep to surface layers of the buccal gingival mucosa (free and attached portions), large collagen fibril bundles became smaller and progressively more wavy with decreasing collagen fibril diameter. This gradual diameter decrease did not occur in the hard palate mucosa (free portion, rugae and interrugal regions) where the fibril diameter remained constant. A link between collagen fibril diameter and mechanical function is discussed.

Electromagnetic stimulation on the bone growth using backscattered electron imaging.

The events at the hydroxyapatite implant material/tissue interface following electromagnetic stimulation were studied in the rabbit. Two kinds of hydroxyapatite were used: natural (NA) and synthetic (HA) both with a grain size of <50 microm. Bone defects, artificially created in rabbit tibiae, were filled with the material examined. One group of animals was exposed immediately after surgery and every 12h thereafter to 30-min treatments with electromagnetic fields (PEMFs). A second group was used as a control (untreated). Two and 4 weeks after implantation, animals were sacrificed and bone samples processed for LM, TEM and SEM using a backscatter electron detector for the evaluation of bone growth. This study indicates that HA has more osteoconductivity than NA, and shows that PEMF-treatment results in a benefit in accelerating bone formation at early time periods.

Structural and functional macrophages alterations by ceramics of different composition.

Biomaterials may initiate several and complex biological reactions in host tissues, and the cell-biomaterial interactions can determine the release of mediators including monocytes and lymphocytes chemotactic factors. The present work was aimed to investigate in vitro the macrophage biological reactions of a natural apatite obtained by heat treatment at 400 degrees C of bovine bone, compared to other ceramics usually used for dental and orthopedic applications, using synthetic apatite and three types of alumina as controls. Particles chemotactic activity and powders oxidative burst evidenced no monocyte macrophages sensitivity reaction for natural and synthetic hydroxyapatite powders at great granulometry (> 50 microm); data were confirmed by ultrastructural observations; electron microscopy analysis showed macrophages with the features of healthy cells in the presence of both natural and synthetic apatites while macrophages grown in the presence of alumina seemed to be negatively affected. In conclusion, among all ceramics tested, natural apatite displayed a good compatibility with living cells, being better tolerated than synthetic hydroxyapatite which in turn is better tolerated than alumina.

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.