Predominant Collagen Fiber Orientation Research Articles

Roles of collagen cross-links and osteon collagen/lamellar morphotypes in equine third metacarpals in tension and compression tests.

Many bones experience bending, placing one side in net compression and the other in net tension. Because bone mechanical properties are relatively reduced in tension compared with compression, adaptations are needed to reduce fracture risk. Several toughening mechanisms exist in bone, yet little is known of the influences of secondary osteon collagen/lamellar 'morphotypes' and potential interplay with intermolecular collagen cross-links (CCLs) in prevalent/predominant tension- and compression-loaded regions. Paired third metacarpals (MC3s) from 10 adult horses were prepared for mechanical testing. From one MC3/pair, 5 mm cubes were tested in compression at several mid-shaft locations. From contralateral bones, dumbbell-shaped specimens were tested in tension. Hence, habitual/natural tension- and compression-loaded regions were tested in both modes. Data included: elastic modulus, yield and ultimate strength, and energy absorption (toughness). Fragments of tested specimens were examined for predominant collagen fiber orientation (CFO; representing osteonal and non-osteonal bone), osteon morphotype score (MTS, representing osteonal CFO), mineralization, porosity and other histological characteristics. As a consequence of insufficient material from tension-tested specimens, CCLs were only examined in compression-tested specimens (HP, hydroxylysylpyridinoline; LP, lysylpyridinoline; PE, pentosidine). Among CCLs, only LP and HP/LP correlated significantly with mechanical parameters: LP with energy absorption, HP/LP with elastic modulus (both r=0.4). HP/LP showed a trend with energy absorption (r=-0.3, P=0.08). HP/LP more strongly correlated with osteon density and mineralization than CFO or MTS. Predominant CFO more strongly correlated with energy absorption than MTS in both testing modes. In general, CFO was found to be relatively prominent in affecting regional toughness in these equine MC3s in compression and tension.

Ultraviolet light induces mechanical and structural changes in full thickness human skin

While the detrimental health effects of prolonged ultraviolet (UV) irradiation on skin health have been widely accepted, the biomechanical process by which photoaging occurs and the relative effects of irradiation with different UV ranges on skin biomechanics have remained relatively unexplored. In this study, the effects of UV-induced photoageing are explored by quantifying the changes in the mechanical properties of full-thickness human skin irradiated with UVA and UVB light for incident dosages up to 1600 J/cm2. Mechanical testing of skin samples excised parallel and perpendicular to the predominant collagen fiber orientation show a rise in the fractional relative difference of elastic modulus, fracture stress, and toughness with increased UV irradiation. These changes become significant with UVA incident dosages of 1200 J/cm2 for samples excised both parallel and perpendicular to the dominant collagen fiber orientation. However, while mechanical changes occur in samples aligned with the collagen orientation at UVB dosages of 1200 J/cm2, statistical differences in samples perpendicular to the collagen orientation emerge only for UVB dosages of 1600 J/cm2. No notable or consistent trend is observed for the fracture strain. Analyses of toughness changes with maximum absorbed dosage reveals that no one UV range is more impactful in inducing mechanical property changes, but rather these changes scale with maximum absorbed energy. Evaluation of the structural characteristics of collagen further reveals an increase in collagen fiber bundle density with UV irradiation, but not collagen tortuosity, potentially linking mechanical changes to altered microstructure.

Collagen fiber orientation pattern, osteon morphology and distribution, and presence of laminar histology do not distinguish torsion from bending in bat and pigeon wing bones.

Bone can adapt to its habitual load history at various levels of its hierarchical structural and material organization. However, it is unclear how strongly a bone's structural characteristics (e.g. cross-sectional shape) are linked to microstructural characteristics (e.g. distributions of osteons and their vascular canals) or ultrastructural characteristics [e.g. patterns of predominant collagen fiber orientation (CFO)]. We compared the cross-sectional geometry, microstructure and ultrastructure of pigeon (Columba livia domestica) humeri, and third metacarpals (B3M) and humeri of a large bat (Pteropus poliocephalus). The pigeon humerus is habitually torsionally loaded, and has unremodeled ('primary') bone with vessels (secondary osteons are absent) and high 'laminarity' because a large majority of these vessels course circularly with respect to the bone's external surface. In vivo data show that the bat humerus is also habitually torsionally loaded; this contrasts with habitual single-plane bending of the B3M, where invivo data show that it oscillates back and forth in the same direction. In contrast to pigeon humeri where laminar bone is present, the primary tissue of these bat bones is largely avascular, but secondary osteons are present and are usually in the deeper cortex. Nevertheless, the load history of humeri of both species is prevalent/predominant torsion, producing diffusely distributed shear stresses throughout the cross-section. We tested the hypothesis that despite microstructural/osteonal differences in these pigeon and bat bones, they will have similar characteristics at the ultrastructural level that adapt each bone for its load history. We postulate that predominant CFO is this characteristic. However, even though data reported in prior studies of bones of non-flying mammals suggest that CFO would show regional variations in accordance with the habitual 'tension regions' and 'compression regions' in the direction of unidirectional habitual bending, we hypothesized that alternating directions of bending within the same plane would obviate these regional/site-specific adaptations in the B3M. Similarly, but for other reasons, we did not expect regional variations in CFO in the habitually torsionally loaded bat and pigeon humeri because uniformly oblique-to-transverse CFO is the adaptation expected for the diffusely distributed shear stresses produced by torsion/multidirectional loads. We analyzed transverse sections from mid-diaphyses of adult bones for CFO, secondary osteon characteristics (size, shape and population density), cortical thickness in quadrants of the cortex, and additional measures of cross-sectional geometry, including the degree of circular shape that can help distinguish habitual torsion from bending. Results showed the expected lack of regional CFO differences in quasi-circular shaped, and torsionally loaded, pigeon and bat humeri. As expected, the B3M also lacked CFO variations between the opposing cortices along the plane of bending, and the quasi-elliptical cross-sectional shape and regional microstructural/osteonal variations expected for bending were not found. These findings in the B3M show that uniformity in CFO does not always reflect habitual torsional loads. Osteon morphology and distribution, and presence of laminar histology also do not distinguish torsion from bending in these bat and pigeon wing bones.

Utility of osteon circularity for determining species and interpreting load history in primates and nonprimates.

Histomorphological analyses of bones are used to estimate an individual's chronological age, interpret a bone's load history, and differentiate species. Among various histomorphological characteristics that can influence mechanical properties of cortical bone, secondary osteon (Haversian system) population density and predominant collagen fiber orientation are particularly important. Cross-sectional shape characteristics of secondary osteons (On.Cr = osteon circularity, On.El = osteon ellipticality) are considered helpful in these contexts, but more robust proof is needed. We sought to determine if variations in osteon shape characteristics are sufficient for accurately differentiating species, load-complexity categories, and regional habitual strain-mode distributions (e.g., tension vs. compression regions). Circularly polarized light images were obtained from 100-micron transverse sections from diaphyses of adult deer calcanei; sheep calcanei, radii, and tibiae; equine calcanei, radii, and third metacarpals (MC3s); chimpanzee femora; and human femora and fibulae. Osteon cross-sectional area (On.Ar), On.Cr, and On.El were quantified indiscriminately and in the contexts of load-complexity and regional strain-mode distributions. On.Cr and On.El, when examined independently in terms of all data, or mean (nested) data, for each bone, exceeded 80% accuracy in the inter-species comparisons only with respect to distinguishing humans from nonhumans. Correct classification among the nonhuman species was <70%. When On.Cr and On.El were coupled together and with On.Ar in discriminant function analyses (nested and unnested data) there were high misclassifications in all but human vs. nonhuman comparisons. Frequent misclassifications in nonhuman comparisons might reflect influences of habitual load complexity and/or strain-mode distributions, or other factors not accounted for by these two considerations.

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.

Interpreting cortical bone adaptation and load history by quantifying osteon morphotypes in circularly polarized light images

Birefringence variations in circularly polarized light (CPL) images of thin plane-parallel sections of cortical bone can be used to quantify regional differences in predominant collagen fiber orientation (CFO). Using CPL images of equine third metacarpals (MC3s), R.B. Martin, V.A. Gibson, S.M. Stover, J.C. Gibeling, and L.V. Griffin. (40) described six secondary osteon variants (‘morphotypes’) and suggested that differences in their regional prevalence affect fatigue resistance and toughness. They devised a numerical osteon morphotype score (MTS) for quantifying regional differences in osteon morphotypes. We have observed that a modification of this score could significantly improve its use for interpreting load history. We hypothesized that our modified osteon MTS would more accurately reveal differences in osteon MTSs between opposing “tension” and “compression” cortices of diaphyses of habitually bent bones. This was tested using CPL images in transverse sections of calcanei from sheep, deer, and horses, and radii from sheep and horses. Equine MC3s and sheep tibiae were examined as controls because they experience comparatively greater load complexity that, because of increased prevalence of torsion/shear, would not require regional mechanical enhancements provided by different osteon morphotypes. Predominant CFO, which can reliably reflect adaptation for a regionally prevalent strain mode, was quantified as mean gray levels from birefringence of entire images (excluding pore spaces) in anterior, posterior, medial, and lateral cortices. Results showed that, in contrast to the original scoring scheme of Martin et al., the modified scheme revealed significant anterior/posterior differences in osteon MTSs in nearly all “tension/compression” bones ( p < 0.0001), but not in equine MC3s ( p = 0.30) and sheep tibiae ( p = 0.35). Among habitually bent bones, sheep radii were the exception; relatively lower osteon populations and the birefringence of the primary bone contributed to this result. Correlations between osteon MTSs using the scoring scheme of Martin et al. with CFO data from all regions of each bone invariably demonstrated weak-to-moderate negative correlations. This contrasts with typically high positive correlations between modified osteon MTSs and regional CFO. These results show that the modified osteon MTS can be a strong correlate of predominant CFO and of the non-uniform strain distribution produced by habitual bending.

Ontogenetic structural and material variations in ovine calcanei: A model for interpreting bone adaptation

Experimental models are needed for resolving relative influences of genetic, epigenetic, and nonheritable functionally induced (extragenetic) factors in the emergence of developmental adaptations in limb bones of larger mammals. We examined regional/ontogenetic morphologic variations in sheep calcanei, which exhibit marked heterogeneity in structural and material organization by skeletal maturity. Cross-sections and lateral radiographs of an ontogenetic series of domesticated sheep calcanei (fetal to adult) were examined for variations in biomechanically important structural (cortical thickness and trabecular architecture) and material (percent ash and predominant collagen fiber orientation) characteristics. Results showed delayed development of variations in cortical thickness and collagen fiber orientation, which correlate with extragenetic factors, including compression/tension strains of habitual bending in respective dorsal/plantar cortices and load-related thresholds for modeling/remodeling activities. In contrast, the appearance of trabecular arches in utero suggests strong genetic/epigenetic influences. These stark spatial/temporal variations in sheep calcanei provide a compelling model for investigating causal mechanisms that mediate this construction. In view of these findings, it is also suggested that the conventional distinction between genetic and epigenetic factors in limb bone development be expanded into three categories: genetic, epigenetic, and extragenetic factors.

The influence of collagen fiber orientation and other histocompositional characteristics on the mechanical properties of equine cortical bone

This study examined relative influences of predominant collagen fiber orientation (CFO), mineralization (% ash), and other microstructural characteristics on the mechanical properties of equine cortical bone. Using strain-mode-specific (S-M-S) testing (compression testing of bone habitually loaded in compression; tension testing of bone habitually loaded in tension), the relative mechanical importance of CFO and other material characteristics were examined in equine third metacarpals (MC3s). This model was chosen since it had a consistent non-uniform strain distribution estimated by finite element analysis (FEA) near mid-diaphysis of a thoroughbred horse, net tension in the dorsal/lateral cortices and net compression in the palmar/medial cortices. Bone specimens from regions habitually loaded in tension or compression were: (1) tested to failure in both axial compression and tension in order to contrast S-M-S vs non-S-M-S behavior, and (2) analyzed for CFO, % ash, porosity, fractional area of secondary osteonal bone, osteon cross-sectional area, and population densities of secondary osteons and osteocyte lacunae. Multivariate multiple regression analyses revealed that in S-M-S compression testing, CFO most strongly influenced total energy (pre-yield elastic energy plus post-yield plastic energy); in S-M-S tension testing CFO most strongly influenced post-yield energy and total energy. CFO was less important in explaining S-M-S elastic modulus, and yield and ultimate stress. Therefore, in S-M-S loading CFO appears to be important in influencing energy absorption, whereas the other characteristics have a more dominant influence in elastic modulus, pre-yield behavior and strength. These data generally support the hypothesis that differentially affecting S-M-S energy absorption may be an important consequence of regional histocompositional heterogeneity in the equine MC3. Data inconsistent with the hypothesis, including the lack of highly longitudinal collagen in the dorsal-lateral ;tension' region, paradoxical histologic organization in some locations, and lack of significantly improved S-M-S properties in some locations, might reflect the absence of a similar habitual strain distribution in all bones. An alternative strain distribution based on in vivo strain measurements, without FEA, on non-Thoroughbreds showing net compression along the dorsal-palmar axis might be more characteristic of the habitual loading of some of the bones that we examined. In turn, some inconsistencies might also reflect the complex torsion/bending loading regime that the MC3 sustains when the animal undergoes a variety of gaits and activities, which may be representative of only a portion of our animals, again reflecting the possibility that not all of the bones examined had similar habitual loading histories.

Dissociation of mineral and collagen orientations may differentially adapt compact bone for regional loading environments: Results from acoustic velocity measurements in deer calcanei

In limb bone diaphyses, it is hypothesized that collagen and extra-fibrillar mineral are aligned differently in relatively simple loading conditions (e.g., habitual longitudinal compression) when compared to complex or potentially deleterious strain environments (e.g., habitual shear or tension). These putative differences in collagen/mineral organization might be adaptations that enhance toughness and fatigue resistance by controlling the direction of microdamage propagation. This study examined relationships between the non-uniform strain distribution of wild deer calcanei and elastic anisotropy of cortical bone specimens in three preparations: (1) demineralized (collagen only), (2) deproteinized (mineral only), and (3) untreated. Using simulated functional loading, the following strain data were obtained from the dorsal “compression”, plantar “tension”, and medial and lateral (“neutral axis”) cortices of one calcaneus of each of seven pairs: (1) peak strain magnitude, (2) prevalent/predominant strain mode (compression, tension, shear), and (3) principal strain orientation with respect to the bone’s long axis. In the contralateral calcanei, elastic anisotropy ratios (ARs) were calculated using acoustic velocity (longitudinal and transverse) measurements from a pair of orthogonally sliced specimens (representing each of three preparation types) from each cortex. In a separate set of seven adult calcanei, predominant collagen fiber orientation (CFO) was measured using circularly polarized light (CPL) in the four cortical locations. Results showed that, in general, elastic anisotropy was significant in each region, with ARs being significantly different from isotropy (where AR = 1.0). Compared to CFO, mineral orientation more strongly influenced this anisotropy, which was most notable in the plantar “tension” cortex. High correlations ( r values from −0.675 to −0.734, P < 0.05) were found between collagen anisotropy obtained from acoustic data when compared to the CPL data. Significant correlations of mineral and collagen anisotropy were also found between strain mode, magnitude, and orientation (all r values ∼−0.750). The habitual compression, tension, and shear (neutral axis) regions also had different collagen/mineral organizations, which may be important in accommodating the well-known disparity in the mechanical properties of bone in these loading modes.

Spatial Distribution of Osteocyte Lacunae in Equine Radii and Third Metacarpals: Considerations for Cellular Communication, Microdamage Detection and Metabolism

Osteocytes, which are embedded in bone matrix, are the most abundant cells in bone. Despite the ideal location of osteocytes to sense the local environment and influence bone remodeling, their functions, and the relative importance of these functions, remain controversial. In this study, we tested several hypotheses that address the possibilities that population densities of osteocyte lacunae (Ot.Lc.N/B.Ar) correlate with strain-, remodeling- or metabolism-related aspects of the local biomechanical environments of mid-third diaphyseal equine radii and third metacarpals from skeletally mature animals. Ot.Lc.N/B.Ar data, quantified in multiple cortical locations, were analyzed for possible correlations with (1) structural and material characteristics (e.g., cortical thickness, percent ash, secondary osteon population density, mean osteon cross-sectional area, and predominant collagen fiber orientation), (2) strain characteristics, including prevalent/predominant strain magnitude and mode (tension, compression, shear), (3) hypothesized strain-mode-related microdamage characteristics, which might be perceived by osteocyte ‘operational’ networks, and (4) variations in remodeling dynamics and/or metabolism (i.e. presumably higher in endocortical regions than in other transcortical locations). Results showed relatively uniform Ot.Lc.N/B.Ar between regions with highly non-uniform strain and strain-related environments and markedly heterogeneous structural and material organization. These results suggest that population densities of these cells are poorly correlated with mechanobiological characteristics, including local variations in metabolic rate and strain magnitude/mode. Although osteocytes hypothetically evolved both as strain sensors and fatigue damage sensors able to direct the removal of damage as needed, the mechanisms that govern the distribution of these cells remain unclear. The results of this study provide little or no evidence that the number of osteocyte lacunae has a functional role in mechanotransduction pathways that are typically considered in bone adaptation.

Ontogenetic and regional morphologic variations in the turkey ulna diaphysis: Implications for functional adaptation of cortical bone

This study examines relationships between bone morphology and mechanically mediated strain/fluid-flow patterns in an avian species. Using mid-diaphyseal transverse sections of domestic turkey ulnae (from 11 subadults and 11 adults), we quantified developmental changes in predominant collagen fiber orientation (CFO), mineral content (%ash), and microstructure in cortical octants or quadrants (i.e., %ash). Geometric parameters were examined using whole mid-diaphyseal cross-sections. The ulna undergoes habitual bending and torsion, and demonstrates nonuniform matrix fluid-flow patterns, and high circumferential strain gradients along the neutral axis (cranial-caudal) region at mid-diaphysis. The current results showed significant porosity differences: 1) greater osteocyte lacuna densities (N.Lac/Ar) (i.e., "non-vascular porosity") in the caudal and cranial cortices in both groups, 2) greater N.Lac/Ar in the pericortex vs. endocortex in mature bones, and 3) greater nonlacunar porosity (i.e., "vascular porosity") in the endocortex vs. pericortex in mature bones. Vascular and nonvascular porosities were not correlated. There were no secondary osteons in subadults. In adults, the highest secondary osteon population densities and lowest %ash occurred in the ventral-caudal, caudal, and cranial cortices, where shear strains, circumferential strain gradients, and fluid displacements are highest. Changes in thickness of the caudal cortex explained the largest proportion of the age-related increase in cranial-caudal breadth; the thickness of other cortices (dorsal, ventral, and cranial) exhibited smaller changes. Only subadult bones exhibited CFO patterns corresponding to habitual tension (ventral) and compression (dorsal). These CFO variations may be adaptations for differential mechanical requirements in "strain-mode-specific" loading. The more uniform oblique-to-transverse CFO patterns in adult bones may represent adaptations for shear strains produced by torsional loading, which is presumably more prevalent in adults. The micro- and ultrastructural heterogeneities may influence strain and fluid-flow dynamics, which are considered proximate signals in bone adaptation.

Relation between collagen fiber orientation and age of osteon formation in human tibial compact bone.

The predominant collagen fiber orientation in newly formed osteons was determined by polarized light microscopy. Osteons were classified into five categories according to the proportion of their cross section formed by dark lamellae. In dark lamellae most fibers run longitudinally or on a step spiral while they follow a circular or shallow spiral course in light lamellae. Types 1 and 2 are 'light' osteons; types 4 and 5 are 'dark', and type 3 is 'intermediate'. A significant correlation was found between the predominant collagen fiber orientation in osteons and the age at which their formation occurs. Significant differences were found also in the collagen fiber orientation in osteons among the different quadrants of the same bone.