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Effect of lacunocanalicular architecture on hydraulic conductance in bone tissue: implications for bone health and evolution.

Bone tissue health depends largely on efficient fluid and solute transport between the blood supply and cells that are the living component of the tissue. We hypothesized that the lacunocanalicular hydraulic network, which is defined by the pericellular fluid space that is common to all bone tissue, is optimized to transport fluid and solutes between the blood supply and bone cells. An analytical study was carried out to evaluate the effect of osteonal architecture, including the osteon diameter, number of annular lamellar regions, and number and length of canalicular channels, on fluid transport between the blood supply and bone cells. On the basis of this analysis, we conclude that osteon size is limited to the distance over which fluid and solutes can be transported efficiently between the blood supply and cells. This analytic model suggests that hydraulic conductivity is highest in lamellar regions closest to the Haversian canal (HC) and decreases with increasing distance from the blood supply, reaching a plateau after the fifth lamella (169 micro m radius). Furthermore, an increase in the diameter of the HC, or a decrease in the length of canaliculi, reduces the hydraulic conductivity within the lacunocanalicular network. Applying the principle of minimal expenditure of energy to this analysis, the path distance comprising five or six lamellar regions represents an effective limit for fluid and solute transport between the blood supply and cells; beyond this threshold, hydraulic resistance in the network increases and additional energy expenditure is necessary for further transportation. This suggests that transport is optimized to meet metabolic demands concomitant with a minimal expenditure of energy. This fundamental new insight into bone structure and physiology may provide a new basis of understanding for tissue engineering, bone physiology in health and disease, and evolutionary biology.

Animals↗

Cement lines of secondary osteons in human bone are not mineral-deficient: new data in a historical perspective.

Using qualitative backscattered electron (BSE) imaging and quantitative energy dispersive X-ray (EDX) spectroscopy, some investigators have concluded that cement (reversal) lines located at the periphery of secondary osteons are poorly mineralized viscous interfaces with respect to surrounding bone. This conclusion contradicts historical observations of apparent highly mineralized (or collagen-deficient) cement lines in microradiographs. Such conclusions, however, may stem from unrecognized artifacts that can occur during scanning electron microscopy. These include specimen degradation due to high-energy beams and the sampling of electron interaction volumes that extend beyond target locations during EDX analysis. This study used quantitative BSE imaging and EDX analysis, each with relatively lower-energy beams, to test the hypothesis that cement lines are poorly mineralized. Undemineralized adult human femoral diaphyses (n = 8) and radial diaphyses (n = 5) were sectioned transversely, embedded in polymethyl methacrylate, and imaged in a scanning electron microscope for BSE and EDX analyses. Unembedded samples were also evaluated. Additional thin embedded samples were stained and evaluated with light microscopy and correlated BSE imaging. BSE analyses showed the consistent presence of a bright line (higher atomic number) coincident with the classical location and description of the cement line. This may represent relative hypermineralization or, alternatively, collagen deficiency with respect to surrounding bone. EDX analyses of cement lines showed either higher Ca content or equivalent Ca content when compared to distant osteonal and interstitial bone. These data reject the hypothesis that cement lines of secondary osteons are poorly mineralized.

Adult↗

Quantitative 3D analysis of the canal network in cortical bone by micro-computed tomography.

Cortical bone is perforated by an interconnected network of porous canals that facilitate the distribution of neurovascular structures throughout the cortex. This network is an integral component of cortical microstructure and, therefore, undergoes continual change throughout life as the cortex is remodeled. To date, the investigation of cortical microstructure, including the canal network, has largely been limited to the two-dimensional (2D) realm due to methodological hurdles. Thanks to continuing improvements in scan resolution, micro-computed tomography (muCT) is the first nondestructive imaging technology capable of resolving cortical canals. Like its application to trabecular bone, muCT provides an efficient means of quantifying aspects of 3D architecture of the canal network. Our aim here is to introduce the use of muCT for this application by providing examples, discussing some of the parameters that can be acquired, and relating these to research applications. Although several parameters developed for the analysis of trabecular microstructure are suitable for the analysis of cortical porosity, the algorithm used to estimate connectivity is not. We adapt existing algorithms based on skeletonization for this task. We believe that 3D analysis of the dimensions and architecture of the canal network will provide novel information relevant to many aspects of bone biology. For example, parameters related to the size, spacing, and volume of the canals may be particularly useful for investigation of the mechanical properties of bone. Alternatively, parameters describing the 3D architecture of the canal network, such as connectivity between the canals, may provide a means of evaluating cumulative remodeling related change.

Haversian System↗

Microelectrode studies of stress-generated potentials in four-point bending of bone.

A microelectrode technique has been developed to enable the study of stress-generated potentials (SGP) in bone to a spatial resolution of 5 micrometers. The technique has been used to measure the electrical potentials as a function of bone micromorphology in four-point bending. Electric fields ranging from 30 to 10(3) times greater than is measured by conventional macroscopic methods have been discovered at the Haversian canals for human and bovine cortical bone. The amplitude and direction of the electric field in the osteons depend specifically upon the amplitude and the sign (i.e., compression or tension) of the stress. The implications of this finding with regard to the origin of SGP and their possible physiological significance are considered.

Animals↗

Microelectrode study of stress-generated potentials obtained from uniform and nonuniform compression of human bone.

By use of a previously developed microelectrode technique, the effect of nonuniform stresses on the stress-generated potentials (SGP) in bone were studied to a resolution of 5 micrometers. Comparison was made between uniformly and nonuniformly applied compression of human cortical bone. It was found that the radial electric fields for osteons in a specimen under uniform compression were equivalent, and such specimens possessed no macroscopic SGP; for nonuniform compression, the electric fields of osteons differed, and a macroscopic SGP was measured. The magnitude of the macroscopic SGP thus appears to be dependent upon local stress differences and, hence, on the SGP of local regions.

Bone and Bones↗

Haversian osteons: longitudinal variation of internal structure.

Previous studies have suggested that one general type of osteon exists, rather than the three types previously classified as light, intermediate, and dark. A method is presented that permits rapid, quantitative measurement of the percent transmission of osteons seen in thin section between crossed polarizers. In studies of osteon segments from human cortical bone, cut either 200 or 5 micrometers in length, a continuous variation of transmission is seen, both for the populations as a whole and for serial sections. These variations cannot be ascribed to angulation between the direction of observation and the axis of the osteonal segment. No correlation can be observed between the angle of the segment axis and the percent transmission. It is suggested that the collagen fiber orientation in osteonal segments must be considered to be continuous, as judged by scattering of polarized light.

Collagen↗

A theory of fatigue damage accumulation and repair in cortical bone.

An analysis is presented of the balance between the accumulation and repair of fatigue damage in osteonal bone. Fatigue damage is defined in terms of cracks seen histologically when precautions are taken to avoid preparation artifact. The rate of occurrence of such damage is assumed to be proportional to the product of applied peak-to-peak stress, raised to a power, and the loading frequency. The rate of damage repair is assumed to be proportional to the activation rate for osteonal remodeling, and to the mean cross-sectional area of the resulting osteons. An additional factor is introduced to account for the possibility that damage provokes nearby remodeling. The theory is used to compare data from previous experiments of two types: fatigue-to-failure, and studies in which histologically observable cracks are made more numerous by repetitive loading. The analysis shows that there is a measure of agreement between the results of the two kinds of experiments, but the current data are too limited, and the results are too dependent on the mode of loading, to adequately test the theory. However, the analysis provides a framework for designing experiments to more efficiently clarify the relationships between fatigue failure, cracks seen in histologic sections, and the rate at which such cracks are repaired by osteonal remodeling.

Biomechanical Phenomena↗

Interactions between microstructural and geometrical adaptation in human cortical bone.

With aging and in disease, the changes in bone microstructure and geometry influence the mechanical properties of cortical bone, however, the level of interaction between the two is not known. Here, we investigate the interaction between the changes in microstructural and geometrical properties of the aging male tibia in proximal and distal middiaphysis. The microstructural measurements include variables related to the size and density of osteons and intracortical porosity. The macroscopic geometrical properties include variables related to bone surfaces (periosteal and endosteal) and cross section (area, moment of inertia). Site-specific correlations were found between the microstructural and geometrical properties along the bone length and at different bone surfaces. In contrast to the proximal middiaphysis of male tibia, where no correlation existed, significant (p<0.05) correlations were found in the distal middiaphysis of tibia. The changes in parameters partially related to bone formation in the cortex, including the osteonal area, showed positive correlations with an increase in the periosteal diameter. Similarly, parameters related to bone resorption and/or failed formation in the cortex, including porosity and pore size, showed significant correlations with cortical thinning. These findings support the concept that, with aging, anabolic and catabolic responses in the human tibia at microstructural and macrostructural levels are spatially related and site specific.

Adult↗

Photoacoustic injury and bone healing following 193nm excimer laser ablation.

The argon-fluoride excimer laser was investigated as a cutting-ablating tool for bone surgery. A total of 52 rats were divided into two experimental groups and two control groups. In one experimental group cortical bone defects were made; in another experimental group defects penetrating into the medullary space were performed. In the two control groups similar defects were achieved using water-cooled carbide burs. The rats were sacrificed on each of the 3, 7, 10, 20, 30, and 40 postoperative day. The cortical bone, the medullary space, and the extrabony tissue were examined by means of light microscopy. In both experimental groups, bone damage, represented by osteocyte destruction, extended to 1,050-1,450 microns ahead from the irradiated site, and bone healing was very much impaired. In the control groups no histological changes could be identified and bone healing appeared to be within normal limits. We believe this extensive bone damage, following 193 nm irradiation, to be a result of photoacoustic waves propagating in the bone following each pulse. In view of our results we feel that excimer lasers presently in use are not suitable for bone surgery. This problem of photoacoustic damage can be overcome in one of two ways: by designing a CW excimer laser or by reducing the pulse width to the picosecond regime.

Animals↗

Use of revascularized periosteal allografts for repairing bony defects: an experimental study.

This is an experimental study on the potential of revascularized periosteal allografts for repair of bony defects. Beijing long-eared white rabbits were used as donors and immunologically incompatible Chinchilla rabbits were used as recipients. The periosteum was stripped off from the femurs of the 6 donor rabbits and transplanted to bony defects of 1.2 cm over the radius of the 12 recipients using microsurgical anastomoses of the blood vessels. In the control group of 8 rabbits the same radial bony defects were produced without the transplantation of periosteal grafts. Immunosuppressants were administered after surgery. The animals were observed for 4 months using a series of radiography, histomorphology, fluorochrome labelling and electron microscopy. Results revealed no bony healing of the defects in the control group, but 9 of the 12 defects healed in the experimental group. The results indicated revascularized periosteal allografts were able to form new bone and heal the 1.2 cm radial bone defect in rabbits.

Anastomosis, Surgical↗

Mathematical modeling of human secondary osteons.

This investigation explores the structural dimensions and patterns within single secondary osteons, with consideration of their biological variation. New data from images obtained previously of osteons observed through linearly polarized light, electron microscopy, and micro-x-ray, combined with recent findings on lamellae by circularly polarized light, confocal microscopy, synchrotron x-ray diffraction, and micro-x-ray, provide the basis for novel computerized models of single osteons and single lamellae. The novelty of such models is the concurrent representation of (1) collagen-hydroxyapatite orientation, (2) relative hydroxyapatite percentage, (3) distributions of osteocytes' lacunae and canaliculae, and (4) biological variations in dimensions of the relevant structures. The mathematical software Maple realizes the computerized models. While the parts of the models are constructed on a personal computer, the voluminous data associated with the representation of lacunar and canalicular distributions require a supercomputer for assembly of the models and final analysis. The programming used to define the models affords the option to randomize the dimensional specifications of osteons, lamellae, lacunae, and canaliculae within the experimentally observed numeric ranges and distributions. Through this option, the program can operate so that each run of the file produces a unique random model within the observed biological variations. The program can also be run to implement specific dimensional requirements. The modeling has applications in the microstructural study of fracture propagation and remodeling, as well as in the simulation of mechanical testing. The approach taken here is of wide application and could be of value in other areas of microscopy such as scanning electron microscopy, microcomputerized tomography scan, and magnetic resonance imaging on cancellous bone structures.

Collagen↗

The effect of alendronate on resorption of the alveolar bone following tooth extraction.

Maintenance of alveolar bone width and height following tooth loss is essential with regard to the restoration of missing teeth with endosseous dental implants or prosthodontics approaches. A various amount of alveolar ridge resorption is likely to occur after tooth extraction at the buccal and lingual alveolar bone plates. Bisphosphonates, alendronate, is well known for its potent inhibition of osteoclast-mediated bone resorption. The objective of this study was to examine the inhibitory effect of alendronate on alveolar bone resorption following tooth extraction in rats. Male Wistar Albino rats were divided into three groups: baseline group, saline-treated group and alendronate-treated group. The saline-treated group was administered with daily saline solution for 2 and 4 weeks respectively while the alendronate-treated group was given a daily amount of 0.25 mg/kg alendronate subcutaneously for the same periods. The level of urinary calcium, creatinine, and serum calcium, alkaline phosphatase and phosphate were measured. Serum alkaline phosphatase level was measured as a marker of osteoblastic activity. Histopathological sections of 4 microm thickness were obtained from the right first mandibular molar region in a bucco-lingual direction. The number of osteoclasts, osteoblasts, and haversian canals, the number and size of resorptive lacunae, and osteoid formation were evaluated histopathologically. The mean thickness of buccal and lingual alveolar bone was measured. In the alendronate-treated group, both buccal and lingual alveolar bone volume reduction was significantly less than the saline treated group. Significant reduction in serum and urinary calcium levels and the number of osteoclasts revealed the pronounced suppression of bone resorption in the alendronate-treated group.

Alendronate↗

Studies on bone marrow histogenesis: morphometric and autoradiographic studies of regeneration marrow stroma in extramedullary autoimplants and after evacuation of marrow cavity.

The marrow cavity of the rat tibia was mechanically evacuated and autoimplanted to the subcutaneous tissue. The regenerative process which restored the integrity of marrow stroma and hemopoiesis, was morphometrically evaluated in whole mount of tibia. Following evacuation, the clot filled the cavity. The granulation tissue then appeared and expanded, penetrating and replacing the clot. The fibroblasts of the granulation tissue differentiated into osteoblasts forming osteoid bone. Within its interstices, the primordial marrow consisting of loose connective tissue and vascular sinuses appeared and hemopoiesis resumed. Expansion of hemopoiesis resulted in the resorption of bone and within three weeks the tibial cavity was restored to the pre-evacuation state. Autoradiography indicated that the labeling index was initially high in fibroblasts and osteoblasts but was subsequently reduced while it increased in osteocytes, cells of Haversian canals, stromal and hemopoietic cells of marrow. The finding is in disagreement with the view that the regenerative process originates from the Haversian canal. When the label was introduced on day 4 post-operatively, it subsequently appeared in osteocytes, cells of Haversian canal, stromal elements of the marrow, but not in the hemopoietic cells. This indicates complete dissociation of marrow stroma and hemopoietic stem cell.

Animals↗

FTIR microspectroscopic analysis of human osteonal bone.

Fourier Transform Infrared Microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of nonosteoporotic human osteonal bone. Spectral maps in four orthogonal directions (in 10 microm steps) from the centers towards the peripheries of individual osteons were obtained from iliac crest biopsies of two necropsy cases. Mineral to matrix ratios, calculated from the ratio of integrated areas of the phosphate nu1,nu3 band at 900-1200 cm-1 to the amide I band at 1585-1725 cm-1, increased from the center to the periphery of the osteon. The total carbonate (based on the nu2 band at approximately 850-900 cm-1) to phosphate nu1,nu3 ratio decreased as the mineral to matrix ratio increased. Analysis of the nu2 CO32- band with a combination of second-derivative spectroscopy and curve fitting revealed a decrease in "labile" carbonate, a slight decrease in Type A and a slight increase in Type B carbonate from the center to the periphery of the osteon. Similar analysis of the components of the nu1,nu3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. These components were assigned by comparison with published frequencies for apatite and acid-phosphate containing calcium phosphates. The most consistent variations were alterations in the relative percent areas of bands at approximately 1020 and approximately 1030 cm-1, which had previously been assigned to nonstoichiometric and stoichiometric apatites, respectively. This ratio was used as an index of variation in crystal perfection throughout the osteon. This ratio decreased as the mineral to matrix ratio increased. The reproducibility of these parameters at multiple sites in multiple biopsies suggests their applicability for the analysis of mineral changes in disease.

Aged↗