A multi-layered poroelastic slab model under cyclic loading for a single osteon.

BACKGROUND: An osteon consists of a multi-layered bone matrix and interstitial fluid flow in the lacunar-canalicular system. Loading-induced interstitial fluid flow in the lacunar-canalicular system is critical for osteocyte mechanotransduction and bone remodelling. METHODS: To investigate the effects of the lamellar structure and heterogeneous material properties of the osteon on the distributions of interstitial fluid flow and seepage velocity, an osteon is idealized as a hollow two-dimensional poroelastic multi-layered slab model subjected to cyclic loading. Based on poroelastic theory, the analytical solutions of interstitial fluid pressure and seepage velocity in lacunar-canalicular pores were obtained. RESULTS: The results show that strain magnitude has a greater influence on interstitial fluid pressure than loading frequency. Interestingly, the heterogeneous distribution of permeability produces remarkable variations in interstitial fluid pressure and seepage velocity in the cross-section of cortical bone. In addition, interstitial fluid flow stimuli to osteocytes are mostly controlled by the value of permeability at the surface of the osteon rather than at the inner wall of the osteon. CONCLUSION: Interstitial fluid flow induced by cycling loading stimuli to an osteocyte housed in a lacunar-canalicular pore is not only correlated with strain amplitude and loading frequency, but also closely correlated with the spatial gradient distribution of permeability. This model can help us better understand the fluid flow stimuli to osteocytes during bone remodelling.

Diversity in intracortical remodeling in the human femoral bone: A novel view point with the morphological analysis of secondary osteons.

INTRODUCTION: In humans, intracortical bone remodeling is performed by a basic multicellular unit (BMU) composed of osteoclasts and osteoblasts penetrating through cortical bones. As a result, secondary osteons and their boundaries, cement lines, can be observed on the transverse section. There have been few reports mention whether there is diversity within a single individual and on the relevance to bone remodeling. The purpose of this study is to investigate the morphological diversity of secondary osteons in human femoral bone and to examine the relationship with bone remodeling. MATERIAL AND METHODS: First of all, we developed an original method to get the cross-sectional images of the cortical bones around the whole circumference for the purpose of evaluating the morphology of the secondary osteon exhaustively. Then, a total of ten cross-sectional slices from one right human femoral bone of male were prepared and stained with this method. The osteon population density and complexity of cement lines in osteons were evaluated in detail. RESULTS: Within this femoral bone, the osteon population density was significantly higher in the periosteal side and in the posterior area. Conversely, the cement line density and the osteon complexity were higher in the endosteal side; the proportion of complexed osteon significantly increased from the periosteal side toward the endosteal side. DISCUSSION: The results suggested that there were diversities in osteon population densities and osteon morphological pattern within one human femoral bone. It seemed that the BMUs ran to avoid the existing regions of osteon in the periosteal sides and to overlap the existing osteon in the endosteal sides. This seemed to be one of the novel viewpoints in the morphological analysis of secondary osteons. It might be better for the orthopedic surgeons to be aware that the osteon distribution in the cortical bone is not uniform.

Coalignment of osteocyte canaliculi and collagen fibers in human osteonal bone.

During bone formation osteocytes get connected with each other via a dense network of canaliculi within the mineralized bone matrix. Important functions attributed to the osteocyte network include the control of bone remodeling and a contribution to mineral homeostasis. To detect structural clues of the formation and functionality of the network, this study analyzes the structure and orientation of the osteocyte lacuno-canalicular network (OLCN), specifically in relation to the concentric bone lamellae within human osteons. The network structure within 49 osteons from four samples of cortical bone from the femoral midshaft of middle-aged healthy women was determined by a combination of rhodamine staining and confocal laser scanning microscopy followed by computational image analysis. A quantitative evaluation showed that 64±1% of the canalicular length has an angle smaller than 30° to the direction towards the osteon center, while the lateral network - defined by an orientation angle larger than 60° - comprises 16±1%. With the same spatial periodicity as the bone lamellae, both radial and lateral network show variations in the network density and order. However, only the preferred orientation of the lateral network twists when crossing a lamella. This twist agrees with the preferred orientation of the fibrous collagen matrix. The chirality of the twist was found to be individual-specific. The coalignment between network and matrix extends to the orientation of the elongated osteocyte lacunae. The intimate link between OLCN and collagen matrix implies an interplay between osteocyte processes and the arrangement of the surrounding collagen fibers during osteoid formation.

A model of osteoblast-osteocyte kinetics in the development of secondary osteons in rabbits.

The kinetics of osteogenic cells within secondary osteons have been examined within a 2-D model. The linear osteoblast density of the osteons and the osteocyte lacunae density were compared with other endosteal lamellar systems of different geometries. The cell density was significantly greater in the endosteal appositional zone and was always flatter than the central osteonal canals. Fully structured osteons compared with early structuring (cutting cones) did not show any significant differences in density. The osteoblast density may remain constant because some of them leave the row and become embedded within matrix. The overall shape of the Haversian system represented a geometrical restraint and it was thought to be related to osteoblast-osteocyte transformation. To test this hypothesis of an early differentiation and recruitment of the osteoblast pool which completes the lamellar structure of the osteon, the number and density of osteoblasts and osteocyte lacunae were evaluated. In the central canal area, the mean osteoblast linear density and the osteocyte lacunae planar density were not significantly different among sub-classes (with the exclusion of the osteocyte lacunae of the 300-1000 µm(2) sub-class). The mean number of osteoblasts compared with osteocyte lacunae resulted in significantly higher numbers in the two sub-classes, no significant difference was seen in the two middle sub-classes with the larger canals, and there were significantly lower levels in the smallest central canal sub-class. The TUNEL technique was used to identify the morphological features of apoptosis within osteoblasts. It was found that apoptosis occurred during the late phase of osteon formation but not in osteocytes. This suggests a regulatory role of apoptosis in balancing the osteoblast-osteocyte equilibrium within secondary osteon development. The position of the osteocytic lacunae did not correlate with the lamellar pattern and the lacunae density in osteonal radial sectors was not significantly different. These findings support the hypothesis of an early differentiation of the osteoblast pool and the independence of the fibrillar lamellation from osteoblast-osteocyte transformation.

Bisphosphonate binding affinity affects drug distribution in both intracortical and trabecular bone of rabbits.

Differences in the binding affinities of bisphosphonates for bone mineral have been proposed to determine their localizations and duration of action within bone. The main objective of this study was to test the hypothesis that mineral binding affinity affects bisphosphonate distribution at the basic multicellular unit (BMU) level within both cortical and cancellous bone. To accomplish this objective, skeletally mature female rabbits (n = 8) were injected simultaneously with both low- and high-affinity bisphosphonate analogs bound to different fluorophores. Skeletal distribution was assessed in the rib, tibia, and vertebra using confocal microscopy. The staining intensity ratio between osteocytes contained within the cement line of newly formed rib osteons or within the reversal line of hemiosteons in vertebral trabeculae compared to osteocytes outside the cement/reversal line was greater for the high-affinity compared to the low-affinity compound. This indicates that the low-affinity compound distributes more equally across the cement/reversal line compared to a high-affinity compound, which concentrates mostly near surfaces. These data, from an animal model that undergoes intracortical remodeling similar to humans, demonstrate that the affinity of bisphosphonates for the bone determines the reach of the drugs in both cortical and cancellous bone.

Changes to the cell, tissue and architecture levels in cranial suture synostosis reveal a problem of timing in bone development.

Premature fusion of cranial sutures is a common problem with an incidence of 3-5 per 10,000 live births. Despite progress in understanding molecular/genetic factors affecting suture function, the complex process of premature fusion is still poorly understood. In the present study, corresponding excised segments of nine patent and nine prematurely fused sagittal sutures from infants (age range 3-7 months) with a special emphasis on their hierarchical structural configuration were compared. Cell, tissue and architecture characteristics were analysed by transmitted and polarised light microscopy, 2D-histomorphometry, backscattered electron microscopy and energy-dispersive-x-ray analyses. Apart from wider sutural gaps, patent sutures showed histologically increased new bone formation compared to reduced new bone formation and osseous edges with a more mature structure in the fused portions of the sutures. This pattern was accompanied by a lower osteocyte lacunar density and a higher number of evenly mineralised osteons, reflecting pronounced lamellar bone characteristics along the prematurely fused sutures. In contrast, increases in osteocyte lacunar number and size accompanied by mineralisation heterogeneity and randomly oriented collagen fibres predominantly signified woven bone characteristics in patent, still growing suture segments. The already established woven-to-lamellar bone transition provides evidence of advanced bone development in synostotic sutures. Since structural and compositional features of prematurely fused sutures did not show signs of pathological/defective ossification processes, this supports the theory of a normal ossification process in suture synostosis - just locally commencing too early. These histomorphological findings may provide the basis for a better understanding of the pathomechanism of craniosynostosis, and for future strategies to predict suture fusion and to determine surgical intervention.

Analysis of osteon morphotype scoring schemes for interpreting load history: evaluation in the chimpanzee femur.

Osteon morphotype scores (MTSs) allow for quantification of mechanically important collagen/lamellar variations between secondary osteons when viewed in circularly polarized ight (CPL). We recently modified the 6-point MTS method of Martin et al. (Martin RB, Gibson VA, Stover SM, Gibeling JC, Griffin LV (1996a) Osteonal structure in the equine third metacarpus. Bone 19, 165-71) and reported superiority of this modified method in correlating with 'tension' and 'compression' cortices of both chimpanzee proximal femoral diaphyses and diaphyses of other non-anthropoid bones that are loaded in habitual bending (Skedros et al. 2009, 2011). In these studies, the 'tension' and 'compression' cortices differed significantly in predominant collagen fiber orientation (CFO) based on weighted-mean gray levels (CFO/WMGLs) in CPL images. In chimpanzee femora, however, some osteons were difficult to score with the 6-point method; namely, 'hybrids' with peripherally bright 'hoops' and variability in alternating rings within the osteon wall. We hypothesized that some of these hybrids would be more prevalent in regions subject to torsion than bending. In this perspective the present study was aimed at expanding our 6-point scoring method (S-6-MTS) into two 12-point methods with six additional morphotypes that considered these hybrids. Three- and 4-point methods were also evaluated. We hypothesized that at least one of these other methods would out-perform the S-6-MTS in terms of accuracy, reliability, and interpreting torsion vs. bending load histories. Osteon morphotypes were quantified in CPL images from transverse sections of eight adult chimpanzee femora (neck, proximal diaphysis, mid-diaphysis), where the mid-diaphysis and base- and mid-neck locations have relatively more complex loading (e.g. torsion + bending) than the proximal diaphysis, where bending predominates. Correlation coefficients between CFO/WMGL and MTSs showed that the S-6-MTS method was either stronger or equivalent to the 12-point methods, and typically stronger than the 3- and 4-point methods for all load environments. In nearly all instances the S-6-MTS is more reliable and accurate when it is applied to cases where interpreting load history requires distinguishing habitual bending from torsion. Consequently, in studies of osteonal adaptations for these load histories the 3- and 4-point methods are not stronger correlates, and the extra time required to assign additional scores in the 12-point methods is both unnecessary and can be highly unreliable.

A weighted osteon morphotype score outperforms regional osteon percent prevalence calculations for interpreting cortical bone adaptation.

Using circularly polarized light microscopy,we described a weighted-scoring method for quantifying regional distributions of six secondary osteon morphotypes(Skedros et al.: Bone 44 (2009) 392-403). This osteon morphotype score (MTS) strongly correlated with "tension" and "compression" cortices produced by habitual bending. In the present study, we hypothesized that the osteon MTS is superior to a relatively simpler method based on the percent prevalence (PP) of these osteon morphotypes. This was tested in proximal femoral diaphyses of adult chimpanzees and habitually bent bones: calcanei from sheep, deer, and horses, radii from sheep and horses, and third metacarpals (MC3s) from horses. Sheep tibiae were examined because their comparatively greater torsion/shear would not require regional variations in osteon morphotypes. Predominant collagen fiber orientation (CFO), a predictor of regionally prevalent/predominant strain mode, was quantified as image gray levels (birefringence). Ten PP calculations were conducted. Although PP calculations were similar to the osteon MTS in corroborating CFO differences between "tension" and "compression" cortices of the chimpanzee femora and most of the habitually bent bones, PP calculations failed to show a compression/tension difference in equine MC3s and sheep radii. With the exception of the prevalence of the "distributed" osteon morphotype, correlations of PP calculations with CFO were weak and/or negative. By contrast, the osteon MTS consistently showed positive correlations with predominant CFO. Compared with the osteon MTS and predominant CFO, regional variations in PP of osteon morpho types are not stronger predictors of nonuniform strain distributions produced by bending.

The shape modulation of osteoblast-osteocyte transformation and its correlation with the fibrillar organization in secondary osteons: a SEM study employing the graded osmic maceration technique.

Cortex fractured surface and graded osmic maceration techniques were used to study the secretory activity of osteoblasts, the transformation of osteoblast to osteocytes, and the structural organization of the matrix around the cells with scanning electron microscopy (SEM). A specialized membrane differentiation at the base of the cell was observed with finger-like, flattened processes which formed a diffuse meshwork. These findings suggested that this membrane differentiation below the cells had not only functioned in transporting collagen through the membrane but also in orienting the fibrils once assembled. Thin ramifications arose from the large and flat membrane foldings oriented perpendicular to the plane of the osteoblasts. This meshwork of fine filaments could not be visualized with SEM because they were obscured within the matrix substance. Their 3-D structure, however, should be similar to the canalicular system. The meshwork of large, flattened processes was no more evident in the cells which had completed their transformation into osteocytes.

Experimental determination of the permeability in the lacunar-canalicular porosity of bone.

Permeability of the mineralized bone tissue is a critical element in understanding fluid flow occurring in the lacunar-canalicular porosity (PLC) compartment of bone and its role in bone nutrition and mechanotransduction. However, the estimation of bone permeability at the tissue level is affected by the influence of the vascular porosity in macroscopic samples containing several osteons. In this communication, both analytical and experimental approaches are proposed to estimate the lacunar-canalicular permeability in a single osteon. Data from an experimental stress-relaxation test in a single osteon are used to derive the PLC permeability by curve fitting to theoretical results from a compressible transverse isotropic poroelastic model of a porous annular disk under a ramp loading history (2007, "Compressible and Incompressible Constituents in Anisotropic Poroelasticity: The Problem of Unconfined Compression of a Disk," J. Mech. Phys. Solids, 55, pp. 161-193; 2008, "The Unconfined Compression of a Poroelastic Annular Cylindrical Disk," Mech. Mater., 40(6), pp. 507-523). The PLC tissue intrinsic permeability in the radial direction of the osteon was found to be dependent on the strain rate used and within the range of O(10(-24))-O(10(-25)). The reported values of PLC permeability are in reasonable agreement with previously reported values derived using finite element analysis (FEA) and nanoindentation approaches.

Tissue level mechanical properties of cortical bone in skeletally immature and mature dogs.

OBJECTIVES: The purpose of this study was to quantify the tissue level mechanical properties of cortical bone of skeletally immature (~five-month-old) Beagle dogs and compare them to data from mature dogs measured in a previous study. METHODS: Eight femoral cross sectional specimens (two bone sections / dog) were obtained from four skeletally immature dogs. A pair of calcein bone labels were administered intravenously to the dogs to mark sites of active mineralization prior to euthanasia. Prepared bone specimens were placed in a nano- indenter specimen holder and the previously identified calcein labelled osteons were located. Labelled (n = 128) and neighbouring unlabelled (n = 127) osteons in skeletally immature femurs were examined by instrumented indentation testing. Indents were made to a depth of 500 nm at a loading rate of 10 nm/s. Indentation modulus (IM) and hardness (H) were obtained. RESULTS: The overall IM of the cortical bone in the skeletally mature groups was significantly greater than in the immature group (p = 0.0011), however overall H was not significantly different. The differences between the groups in IM were significant for the unlabelled osteons (p = 0.001), but not for the labelled osteons (p = 0.56). CONCLUSION: There are differences in the IM of unlabelled osteons in skeletally immature and mature groups of Beagle dogs. In contrast to whole bone mechanical tests, where there are obvious differences between growing and mature bones, there are only small differences in the micro-mechanical properties.

Osteoblasts infill irregular pores under curvature and porosity controls: a hypothesis-testing analysis of cell behaviours.

The geometric control of bone tissue growth plays a significant role in bone remodelling, age-related bone loss, and tissue engineering. However, how exactly geometry influences the behaviour of bone-forming cells remains elusive. Geometry modulates cell populations collectively through the evolving space available to the cells, but it may also modulate the individual behaviours of cells. To factor out the collective influence of geometry and gain access to the geometric regulation of individual cell behaviours, we develop a mathematical model of the infilling of cortical bone pores and use it with available experimental data on cortical infilling rates. Testing different possible modes of geometric controls of individual cell behaviours consistent with the experimental data, we find that efficient smoothing of irregular pores only occurs when cell secretory rate is controlled by porosity rather than curvature. This porosity control suggests the convergence of a large scale of intercellular signalling to single bone-forming cells, consistent with that provided by the osteocyte network in response to mechanical stimulus. After validating the mathematical model with the histological record of a real cortical pore infilling, we explore the infilling of a population of randomly generated initial pore shapes. We find that amongst all the geometric regulations considered, the collective influence of curvature on cell crowding is a dominant factor for how fast cortical bone pores infill, and we suggest that the irregularity of cement lines thereby explains some of the variability in double labelling data as well as the overall speed of osteon infilling.

Bone tissue aging affects mineralization of cement lines.

Cement lines are known as thin peripheral boundaries of the osteons. With a thickness below 5 µm their composition of inorganic and organic compounds has been a matter of debate. Here, we hypothesized that cement lines become hypermineralized and their degree of mineralization is not constant but related to the tissue age of the osteon. Therefore, we analyzed the calcium content of osteons and their corresponding cement lines in a range of different tissue ages reflected by osteonal mineralization levels in femoral cortical bone of both postmenopausal women with osteoporosis and bisphosphonate-treated cases. Quantitative backscattered electron imaging (qBEI) showed that cement lines are hypermineralized entities with consistently higher calcium content than their corresponding osteons (mean calcium content: 29.46 ±â€¯0.80 vs. 26.62 ±â€¯1.11 wt%; p < 0.001). Micro-Raman spectroscopy complemented the qBEI data by showing a significantly higher phosphate/amide I ratio in the cement lines compared to the osteonal bone (8.78 ±â€¯0.66 vs. 6.33 ±â€¯0.58, p < 0.001), which was both due to an increased phosphate peak and a reduced amide I peak in cement lines. A clear positive correlation of cement line mineralization and the mineralization of the osteon was observed (r = 0.839, p = 0.003). However, the magnitude of the difference between cement line and osteonal calcium content decreased with increased osteonal calcium content (r = -0.709, p < 0.001), suggesting diverging mineralization dynamics in these osseous entities. The number of mineralized osteocyte lacunae per osteon bone area correlated positively with both osteonal and cement line calcium content (p < 0.01). The degree of mineralization of cement lines may represent another tissue-age related phenomenon, given that it strongly relates to the osteonal mineralization level. Understanding of the cement lines' mineralization and their changes in aging and disease states is important for predicting crack propagation pathways and fracture resistance mechanisms in human cortical bone.

Are distributions of secondary osteon variants useful for interpreting load history in mammalian bones?

BACKGROUND/AIMS: In cortical bone, basic multicellular units (BMUs) produce secondary osteons that mediate adaptations, including variations in their population densities and cross-sectional areas. Additional important BMU-related adaptations might include atypical secondary osteon morphologies (zoned, connected, drifting, elongated, multiple canal). These variants often reflect osteonal branching that enhances toughness by increasing interfacial (cement line) complexity. If these characteristics correlate with strain mode/magnitude-related parameters of habitual loading, then BMUs might produce adaptive differences in unexpected ways. METHODS: We carried out examinations in bones loaded in habitual torsion (horse metacarpals) or bending: sheep, deer, elk, and horse calcanei, and horse radii. Atypical osteons were quantified in backscattered images from anterior, posterior, medial, and lateral cortices. Correlations were determined between atypical osteon densities, densities of all secondary osteons, and associations with habitual strain mode/magnitude or transcortical location. RESULTS: Osteon variants were not consistently associated with 'tension', 'compression', or neutral axis ('shear') regions, even when considering densities or all secondary osteons, or only osteon variants associated with relatively increased interfacial complexity. Similarly, marrow- and strain-magnitude-related associations were not consistent. CONCLUSION: These data do not support the hypothesis that spatial variations in these osteon variants are useful for inferring a habitual bending or torsional load strain history.

Osteon interfacial strength and histomorphometry of equine cortical bone.

The interfacial strength of secondary osteons from the diaphysis of the Thoroughbred equine third metacarpal was evaluated using the fiber pushout test. The pushout was performed on 300-500 microm sections of 4x4x15 mm bone blocks machined from four anatomic regions of the cortex. Pushout strength was evaluated from proximal to distal location within the diaphysis on four osteon types classified under polarized light on adjacent histologic sections from each block. The shear strength of the interfaces were estimated from shear lag theory. Differences were found in the interfacial strength of osteons based on appearance under polarized light with bright field having the highest interfacial strength (40.3 MPa). The lowest strength was found in the dark field osteons (22.8 MPa). The dorsal region had the highest shear strength and toughness compared to all other regions. The cement line and interlamellar interfaces are similar in strength, but exhibit regional dependence--specifically, the palmar region strength is less (17.5 MPa) than the osteon interlamellar interfaces (30.4 MPa) and osteon type dependent (alternating significantly weaker than other types). Histomorphometry revealed significant regional differences (p<0.0001) in osteon area fraction among the four osteon types as well as differences in the osteon diameter (p=0.01), with dorsal regions having larger osteons (170 microm) than the palmar region (151 microm). Fatigue life and fracture toughness of Haversian bone are reported in the literature to be regionally dependent and are known to be associated with osteon pullout--an osteon interfacial phenomenon. Therefore, the results presented in this study are important to further the understanding of the mechanisms of fragility and damage accumulation in cortical bone.

Osteonal effects on elastic modulus and fatigue life in equine bone.

We hypothesized that recently formed, incompletely mineralized, and thus, relatively deformable osteons in the equine third metacarpus enhance in vitro load-controlled fatigue life in two ways. Macroscopically, there is a compliance effect, because reduced tissue elastic modulus diminishes the stress required to reach a given strain. Microscopically, there is a cement line effect, in which new osteons and their cement lines more effectively serve as barriers to crack propagation. We studied 18 4 x 10 x 100 mm beams from the medial, lateral, and dorsal cortices of metacarpal bones from 6 thoroughbred racehorses. Following load-controlled fatigue testing to fracture in 4 point bending, a transverse, 100 microm thick, basic fuchsin-stained cross-section was taken from the load-bearing region. The number and diameter of all intact (and thus recently formed/compliant) secondary osteons in a 3.8 x 3.8 mm region in the center of the section were determined. The associated area fraction and cement line length of intact osteons were calculated, and the relationships between these variables, elastic modulus (E), and the logarithm of fatigue life (logN(F)) were analyzed. As expected, logN(F) was negatively correlated with E, which was in turn negatively correlated with intact osteon area fraction and density. (LogN(F))/E increased in proportion to intact osteon density and nonlinearly with cement line density (mm/mm(2)). These results support the hypothesis that remodeling extends load-controlled fatigue life both through the creation of osteonal barriers to microdamage propagation and modulus reduction.

Micro- and nano-structural analyses of damage in bone.

Skeletal fractures represent a significant medical and economic burden for our society. In the US alone, age-related hip, spine, and wrist fractures accounted for more than $17 billion in direct health care costs in 2001. Moreover, skeletal fractures are not limited to the elderly; stress fractures and impact/trauma-related fractures are a significant problem in younger people also. Gaining insight into the mechanisms of fracture and how these mechanisms are modulated by intrinsic as well as extrinsic factors may improve the ability to define fracture risk and develop and assess preventative therapies for skeletal fractures. Insight into failure mechanisms of bone, particularly at the ultrastructural-level, is facilitated by the development of improved means of defining and measuring tissue quality. Included in these means are microscopic and spectroscopic techniques for the direct observation of crack initiation, crack propagation, and fracture behavior. In this review, we discuss microscopic and spectroscopic techniques, including laser scanning confocal microscopy (LSCM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopic imaging for visually observing microdamage in bone, and the current understanding of damage mechanisms derived from these techniques.

Biological fixation of endosseous implants.

Primary implant stability is ensured by a mechanical fixation of implants. However, during implant healing a biological anchorage is necessary to achieve final osseointegration. Aim of this study was to investigate the histological aspects of biological fixation around titanium screws. Forty-eight titanium screws with different surfaces (smooth, plasma sprayed, sand blasted) were inserted in tibiae and femura of sheep and analyzed by light microscope and SEM 1 hour, 14 and 90 days after implantation. One hour after implantation the implant-bone gap was filled with a blood clot and host bone chips arising from burr surgical preparation or friction during implant insertion. Fourteen days after implantation new trabecular bone and enveloped bone chips were observed in the gap: no osteogenesis developed where implant threads were in contact with host bone. Ninety days after surgery all trabecular bone and most of the bone chips were substituted by a mature lamellar bone with few marrow spaces. Our results suggest that the trabecular bone and bone chips represent a three-dimensional network ensuring a biological implant fixation in all different implant surfaces 2 weeks after surgery. Host bone chips could favour the peri-implant osteogenesis. Inter-trabecular and implant-trabecular marrow spaces of both trabecular and lamellar bone may favour the peri-implant bone turnover.

Peri-implant osteogenesis in health and osteoporosis.

Long-term clinical success of endosseous dental implants is critically related to a wide bone-to-implant direct contact. This condition is called osseointegration and is achieved ensuring a mechanical primary stability to the implant immediately after implantation. Both primary stability and osseointegration are favoured by micro-rough implant surfaces which are obtained by different techniques from titanium implants or coating the titanium with different materials. Host bone drilled cavity is comparable to a common bone wound. In the early bone response to the implant, the first tissue which comes into contact with the implant surface is the blood clot, with particular attention to platelets and fibrin. Peri-implant tissue healing starts with an inflammatory response as the implant is inserted in the bone cavity, but an early afibrillar calcified layer comparable to the lamina limitans or incremental lines in bone is just observable at the implant surface both in vitro than in vivo conditions. Just within the first day from implantation, mesenchymal cells, pre-osteoblasts and osteoblasts adhere to the implant surface covered by the afibrillar calcified layer to produce collagen fibrils of osteoid tissue. Within few days from implantation a woven bone and then a reparative trabecular bone with bone trabeculae delimiting large marrow spaces rich in blood vessels and mesenchymal cells are present at the gap between the implant and the host bone. The peri-implant osteogenesis can proceed from the host bone to the implant surface (distant osteogenesis) and from the implant surface to the host bone (contact osteogenesis) in the so called de novo bone formation. This early bone response to the implant gradually develops into a biological fixation of the device and consists in an early deposition of a newly formed reparative bone just in direct contact with the implant surface. Nowadays, senile and post-menopausal osteoporosis are extremely diffuse in the population and have important consequences on the clinical success of endosseous dental implants. In particular the systemic methabolic and site morphological conditions are not favorable to primary stability, biological fixation and final osseointegration. An early good biological fixation may allow the shortening of time before loading the implant, favouring the clinical procedure of early or immediate implant loading. Trabecular bone in implant biological fixation is gradually substituted by a mature lamellar bone which characterizes the implant ossoeintegration. As a final consideration, the mature lamellar bone observed in osseointegrated implants is not always the same as a biological turnover occurs in the peri-implant bone up to 1mm from the implant surface, with both osteogenesis and bone reabsorption processes.

Bone microarchitecture evaluated by histomorphometry.

The increasing use of densitometric devices for assessing bone fragility has progressively strengthened the assumption that mass is the most important property determining bone mechanical competence. Nevertheless, structure and microarchitecture are relevant aspects of bone strength. The study of microarchitecture is based on the measure of width, number, and separation of trabeculae as well as on their spatial organization. There are several methods to assess bone architecture, particularly at the trabecular level. In particular, histomorphometry, based on the use of optical microscopy and on the principles of quantitative histology and stereology, evaluates microarchitecture two-dimensionally, even if these measures appear well correlated to the three-dimensional structure and properties of bone. In addition, new computerized methods allow the acquisition of more sophisticated measurements by means of a digitizer have been introduced to integrate the use of the microscope. These methods supply information on trabecular width as well as on its distribution and on the organization of the trabeculae in the marrow space. Microarchitecture seems to be a determinant of bone fragility independent of bone density and it is important for understanding the mechanisms of bone fragility as well as the action of the drugs used to prevent osteoporotic fractures. Several in vivo studies (on animals and humans) can provide an additional interpretation for the anti-fracture effect of such drugs. For instance, bisphosphonates and parathyroid hormone seem to preserve or even improve microarchitecture. The challenge for the future will be to evaluate bone quality in vivo with the same or better resolution and accuracy than the invasive methods used today.