The impact of age, mineralization, and collagen orientation on the mechanics of individual osteons from human femurs

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The impact of age, mineralization, and collagen orientation on the mechanics of individual osteons from human femurs Caitlyn J. Collins , Maria Kozyrev , Martin Frank , Orestis G. Andriotis , Ruth A. Byrne , Hans P. Kiener , Michael L. Pretterklieber , Philipp J. Thurner PII: DOI: Reference:

S2589-1529(19)30369-2 https://doi.org/10.1016/j.mtla.2019.100573 MTLA 100573

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Materialia

Received date: Accepted date:

27 September 2019 17 December 2019

Please cite this article as: Caitlyn J. Collins , Maria Kozyrev , Martin Frank , Orestis G. Andriotis , Ruth A. Byrne , Hans P. Kiener , Michael L. Pretterklieber , Philipp J. Thurner , The impact of age, mineralization, and collagen orientation on the mechanics of individual osteons from human femurs, Materialia (2019), doi: https://doi.org/10.1016/j.mtla.2019.100573

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The impact of age, mineralization, and collagen orientation on the mechanics of individual osteons from human femurs Caitlyn J. Collinsa,1,*, Maria Kozyreva,*, Martin Franka, Orestis G. Andriotisa, Ruth A. Byrneb, Hans P. Kienerb, Michael L. Pretterklieberc, Philipp J. Thurnera a

Institute of Lightweight Design and Structural Biomechanics, Vienna University of

Technology, Getreidemarkdt 9, 1060 Vienna, Austria b

Department of Medicine III, Division of Rheumatology, Medical University of Vienna,

Währinger Gürtel 18-20, 1090 Vienna, Austria c

Center for Anatomy and Cell Biology, Division of Anatomy, Medical University of Vienna,

Währinger Straße 13, 1090 Vienna, Austria

Corresponding Author: 1

Caitlyn J. Collins

[email protected] Leopold-Ruzicka-Weg 4, HCP H 11.3 8093 Zurich, Switzerland +41 44 633 9199 *Caitlyn J. Collins and Maria Kozyrev contributed equally to this manuscript

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Abstract Osteons are the end product of cortical bone remodeling. Changes in their mechanical properties may be an indicator of overall bone health. Aszenci and Bonucci first evaluated the compressive properties of individual osteons in 1968. However, these results have never been independently validated and technological advances allow for further investigation of osteon mechanics. In the present study, an experimental protocol was established such that osteons ~250 µm in diameter were successfully extracted from human cortical bone (males, ages 93 and 64) and loaded in compression. Both micro-computed tomography and second harmonic generation (SHG) microscopy imaging methods were incorporated into the protocol to improve the structural and compositional assessment of each osteon. Further, a highresolution videography system was used to monitor the osteons during mechanical testing, permitting insight into deformation mechanisms in situ. The results show that the older donor osteons (male, age 93 (n=13)) were stiffer (p = 0.04) and more highly mineralized (p = 0.03), while the younger donor osteons (male, 64 (n=14)) exhibited more heterogeneity in the measured mechanical properties with the presence of both stiff and more compliant osteons. SHG intensity indicated predominantly longitudinal fiber alignment for all osteons while the videography data revealed three distinct failure modes and confirmed that whitening observed during yielding of macroscale bone specimens represents failure at the local level. The stiffness and strength of the younger donor osteons were heavily dependent on the gross structure of the osteon, while these properties were dominated by collagen orientation in the older donor osteons.

Keywords- Cortical bone, femur, osteon, mechanical properties, failure

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AbbreviationsAw – total, visible osteon surface area BV/TV – bone volume fraction °C – degrees Celsius CNC – computer numeric control CSA – cross-sectional area D – Feret’s diameter DI – deionized E – effective compressive modulus H – height HBSS – Hanks Balanced Salt Solution – maximum grey-value of the ROI ̅

– mean grey-value intensity of the ROI

PBS – phosphate buffer solution ROI – region of interest SHG – second harmonic generation TMD – tissue mineral density µCT – microcomputed tomography WElastic – elastic work WPost-yield – post-yield work εDamageOnset – damage onset strain εmax – maximum strain εyield – yield strain σmax – maximum stress σyield – yield stress

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Statement of Significance Osteons, the functional unit of human cortical bone, are the product of bone remodeling. As such, changes in their mechanical properties may be an indicator of overall bone health. Despite the extensive literature on bone mechanical properties, existing data on individual osteons is limited to the work of Ascenzi and Bonucci who pioneered the extraction and mechanical testing of such structural elements in the 1960s and 70s. The present study reestablishes and modernizes a method for mechanical and compositional characterization of a bone at this under-investigated length scale. The addition of more advanced imaging methods enabled the identification of distinct failure modes and confirmed that whitening observed during yielding of macroscale bone specimens represents failure at the local level.

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1

Introduction Musculoskeletal disorders are estimated to be the second most common cause of

disability worldwide, with an increase in incidence of 45% between 1990 and 2010 [1]. Disability due to a musculoskeletal disorder imposes a great economic burden on the life of the individual as well as on social and economic systems. In 2015 the aggregated cost of musculoskeletal disease took up 5.7 % of the United States’ gross domestic profit (GDP) [2]. Moreover, the increasing incidence of pathological fractures indicates a clear need for an improvement in the current approach to diagnosis, prevention, and treatment of bone diseases [3]. Despite this, challenges in developing new approaches for diagnosis and treatment arise due to poor understanding of the complex structure and mechanobiological behavior of bone. Bone is hierarchically organized and made up of an organic/inorganic matrix that undergoes constant remodeling. Seven hierarchical levels are commonly distinguished [4], ranging from the whole bone level to the nanoscale level. At the nanoscale level, bone is broken into its basic components, collagen, mineral, non-collagenous proteins, and water. While each length scale contributes to the overall strength and toughness [5] of bone, the level or structural element most relevant to cortical bone remodeling is the osteon. Osteons are cylindrical structures that result from the concerted activity of osteoclasts and osteoblasts, which first form and then refill a remodeling cavity. As a result, investigating old and newly formed osteons may reveal “time-lapsed” changes in bone quality, i.e. bone composition and/or structure, associated with the progression of ageing or disease [6–9]. While information on the mechanical properties of osteons is available, the existing data is largely if not solely based on the work of Ascenzi and Bonucci who pioneered the extraction and mechanical testing of such structural elements in the 1960s and 70s [10–14]. Compressive mechanical properties of individual osteons extracted from tissue of a 30year-old donor were first evaluated by Ascenzi and Bonucci in 1968 [11]; the values of compressive moduli were reported to vary from 1.6 to 9.3 GPa, depending on their 5

structural organization and degree of mineralization. Osteons were classified into three structural types, transversal, alternate, and longitudinal, based on their appearance under polarizing light microscopy [10]. The cross-sections of transversal osteons appeared light or bright, the cross-sections of alternate osteons appeared grey, and the cross-sections of longitudinal osteons appeared dark. Ascenzi and Bonucci attributed the differences in appearance to the orientation of the collagen fiber bundles in adjacent lamellae. Light osteons were thought to have collagen fiber bundles with predominantly transverse (0°) orientation in adjacent lamellae while dark osteons were thought to have collagen fiber bundles with predominantly longitudinal (90°) alignment. Grey osteons, therefore, were assumed to have a near cross-ply (0°/90°) orientation of collagen fiber bundles in adjacent lamellae. The exact spatial organization of fiber bundles within adjacent osteonal lamellae remains a topic of debate. While osteon lamellae do appear light or dark under polarized light microscopy, a number of models have been proposed to explain the observed differences. Ascenzi and Bonucci’s model is grouped with those which attribute the difference in appearance to the fibril orientation in adjacent lamellae while contrasting theories propose that the fibrils are homogeneously distributed and, therefore, local fibril density is responsible for the differences in appearance [15]. Significant advances in imaging technologies have been made since Acenzi and Bonucci’s original study. Therefore, incorporating techniques such as x-ray diffraction, second harmonic generation (SHG) microscopy or small angle x-ray scattering may lead to novel discoveries in the relationship between the mechanical properties of individual osteons and collagen fibril orientation. To date, the study by Nyman et al. [9] is the only other reported experiment to excise individual osteons from the mid-diaphysis of femurs of middle age and elderly body donors for crosslinking analysis. In their study, a modified computer-numeric-control (CNC) system

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and an eccentric milling bit were used to core out samples, semi-automating the extraction technique developed by Ascenzi and Bonucci [10]. Direct measurement of the mechanical properties of cortical bone at and below the osteonal level is typically achieved through micromechanical tests or nanoindentation. An elastic modulus of 18.6 ± 3.5 GPa was reported by Rho et al. [16] from microtensile experiments of cortical bone specimens (~0.3 mm in thickness) excised from the diaphyseal region of a human tibia. Similarly, nanoindentation experiments on human cortical bone from the mid-diaphysis of the femur yielded elastic moduli between 17 GPa and 27 GPa, with osteonal lamellae representing the lower end of the spectrum [17–19]. More recently, focused ion beam milling techniques have been used to isolate well-defined micron-sized volumes on the order of individual lamellae. As a result, cyclic micropillar compression tests revealed that dry, ovine osteonal lamellar bone samples with thicknesses in the micrometer range have an elastic modulus of 31 ± 6.5 GPa in the longitudinal direction [20], while rehydrated ovine micropillars were found to have a 20% lower elastic modulus than those of air-dried samples [21]. Notably, the elastic modulus values reported by Ascenzi and Bonucci [11] are well below those from nanoindentation or other micromechanical tests. The experiments conducted by Ascenzi and Bonucci have never been reproduced by an independent laboratory, likely due to experimental challenges. Considering the technological advances over the last 50 years, reproduction of these experiments bears several promises: Firstly, advances made in digital imaging enable the use of high-resolution videography systems for monitoring samples during mechanical testing, permitting insight into deformation mechanisms in situ. Secondly, imaging techniques such as micro-computed tomography (µCT) or SHG microscopy can give quantitative information on mineralization, vascular structures, and collagen orientation within the extracted osteons. Thirdly, the mechanical and structural properties of individual osteons have been underinvestigated. Examining the mechanical behavior and fracture mechanisms of individual 7

osteons could provide essential insights into fracture at the whole bone level and elucidate mechanisms involved in bone ageing since the portion of bone under analysis stems from a specific point in time. In this context, probing the mechanics of individual osteons may help to develop new effective approaches for diagnosis and treatment of bone diseases. To address these opportunities, the major aim of the current study was to reproduce the compression experiments reported by Ascenzi and Bonucci [11] and check the validity of their reported results. To this end, a robust preparation protocol was established in order to excise and analyze individual osteons from cross-sectional slices of human cortical bone. The second aim was to combine mechanical testing with videographic imaging to investigate damage mechanisms and their impact on the mechanical properties of individual osteons.

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Materials and Methods Sample preparation Two unembalmed femurs from human anatomic specimens (males, ages 93 and 64)

were obtained from the Division of Anatomy of the Medical University of Vienna and stored at -80 degrees Celsius (°C). The use of body donor material was approved by the Ethics Committee of the Medical University of Vienna (EK Nr: 1744/2016). A hand saw was used to cut 4 cm sections from the mid-diaphysis of the frozen femurs, approximately 14 cm distal to the femur head (50% of the femur height) (Fig. S.1a). These sections were then cut lengthwise into medial and lateral segments using a band saw with a diamond-coated blade (Exact 300 CP, Norderstedt, Germany). A low speed precision saw (IsoMet, Buehler, Lake Bluff, IL, USA) outfitted with a diamond wafering blade was then used to cut a number of axial cross-sections with thicknesses ranging from 400 µm to 500 µm from these medial and lateral mid-diaphysis segments . Irrigation with deionized (DI) water was used to prevent overheating during the cutting steps. The obtained cross-sections were firmly fixed on a plastic slide using superglue (Super Glue Liquid, Pattex, Germany). To ensure the osteonal structures would be visible with a light microscope, the affixed cross-sections were 8

sequentially hand-polished using 1000, 1200, and 1500 grit sand papers followed by a 3 µm particle size diamond suspension. The cross-sections were rinsed with DI water between each grit sand paper and sonicated in DI water after the final polishing step for 5 minutes in order to remove any remaining debris from the surface. After polishing, the axial crosssections were stored in Hanks Balanced Salt Solution (HBSS, pH 7.4) at -80°C. A CNC machine (BZT PFK-0203-PX, BZT Maschinenbau GmbH, Germany) was used to extract individual osteons from the donor cross-sections. Prior to milling, the medial and lateral cross-sections were removed from the freezer, fixed to the 2-axis moving stage of the CNC by a custom clamping set-up (Fig. S.1b), immersed in a water bath filled with DI water, and positioned under the spindle of the CNC (see Fig. S.2). A camera (Mako U-130B USB 3.0, AlliedVision, USA) with an optical microscope (12X Zoom Lens System, Navitar, USA) was mounted to the CNC in order to provide visual feedback for accurate positioning of the drill bit above the osteonal structures. The video feed was captured using a custom MATLAB (Mathworks, Inc., Natick, Massachusetts, United States) script, which enabled micrometer-precise live positioning (±20 µm). Osteons with a high degree of circularity in both outer and Haversian canal diameter were targeted for extraction. Once a target osteon was identified with the camera, the osteon was milled out with a custom-made stainless steel eccentric drill-bit using a spindle speed of 7000 rpm. After the drilling procedure, osteons were manually extracted from the water-bath and examined with reflected light microscopy at 50x magnification to detect any apparent flaws in osteonal geometry. In a single milling session (< 8 hours), 20 osteons were extracted from the older donor and 18 were extracted from the younger donor. Only the osteons without external imperfections were subjected to further analysis. The final sets of osteons (age 94 (n=13), age 64 (n=14)) were stored in vials of HBSS (pH 7.4) in the freezer at -80°C until imaging.

2.2 Micro-computed Tomography Extracted osteons were submerged in HBSS (pH 7.4) and scanned using µCT (Scanco Medical AG, Bassersdorf, Switzerland) at 70 kV and 145 µA with a voxel resolution of 3.3 µm. Four calibration phantoms of 100, 210, 415 and 790 mg HA/cm3 were scanned using 9

the same settings to determine equation coefficients for density calibration of the µCT data. The µCT data was processed using ImageJ 1.51o and BoneJ 1.4.2 [22] in order to evaluate morphological, compositional, and structural properties of the extracted osteons. The µCT stacks of the osteons were first resliced such that the slice thickness was perpendicular to the transverse cross-section. Haversian canal morphology was assessed for irregularities such as branching or deviations in the canal angle. A greyscale value of 110 and the derived density calibration coefficients were applied to the slice geometry macro of BoneJ in order to determine the following structural and compositional properties of each uCT slice within the reoriented stack: osteon diameter (D), cross-sectional area (CSA), bone volume ratio (BV/TV) and bone tissue mineral density (TMD) (Fig. 1). Feret’s diameter was estimated for each µCT slice through the height of the osteon and averaged to get D. The height (H) of each osteon was manually measured three times by determining the perpendicular distance between the top and the bottom planes of the osteon and then averaged. The cross-sectional area of the osteon from each µCT slice, an annulus defined by the applied threshold, was measured through the height of the osteon and then averaged to get CSA. BV/TV was calculated from a cylindrical region of interest that included the Haversian canal. Low BV/TV values represented a high non-mineral volume fraction of the osteon and, therefore, a large Haversian canal size. TMD was determined using a density calibration equation to convert the mean grey value of the frontal osteonal cross-section to a physical density in mg HA/cm3.

2.3 Second harmonic generation microscopy To assess collagen fibril orientation, a SP5 Leica confocal/multiphoton microscope (SP5 Leica, Leica Microsystems, Mannheim, Germany) equipped with a titanium sapphire infrared laser (Spectra-Physics GmbH, Germany) was used to perform SHG imaging on all osteons. The transverse cross-section of each osteon was imaged in phosphate buffer solution (PBS) at room temperature (10mM, pH7.4) with an 800 nm excitation wavelength. The grey-value intensity of the recorded SHG signal was used to interpret the orientation of

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the collagen fibrils within the transverse plane relative to the long axis of the osteon using Eq. (1). (√ Herein, ̅ image and

̅

)

(1)

is the mean grey-value intensity of the region of interest (ROI) in the SHG is the maximum grey-value intensity of the ROI. Higher grey-value intensity

indicated fibrils were more parallel to the imaged cross-section, i.e. transverse, whereas lower grey-value intensity indicated fibrils were more perpendicular to the cross section, i.e. longitudinal (Fig. 2). The mean and standard deviation of the Collagen Angle for each donor was calculated from the population mean and square root of the variance of the summed distributions from each individual osteon (See Fig. S.3). Spatial variation in the distribution of transverse and longitudinal collagen fibers in adjacent lamellae for sample osteons is given in Fig. S.4.

2.4 Mechanical testing After the morphological, compositional, and structural properties were characterized, harvested osteons were loaded under uniaxial compression within a water bath filled with HBSS (pH 7.4) using a servo-electric axial testing system (SELmini-001, Thelkin AG, Winterthur, Switzerland) (See Fig. S.2). Displacement was applied such that the strain rate was 0.0025 s−1 for each osteon up to a maximum strain of 20%. A high-resolution video camera (UI-3250CP-M- GL, IDS GmbH, Germany) with a telescopic lens system (KITOADP-0.5 adapter, Kitotec GmbH, Germany) was used to track localized displacements and to observe the mode of failure. Failure was defined as the point of maximum force. The camera had a chip size of 1600x1200 pixels and the calibrated pixel edge length was 1.7 µm. Displacement in µm was measured from the videography data using Trackpy v0.3.2 [23] and converted into axial strain. Force was measured directly from the load cell of the servo-electric testing system and converted into a compressive stress using Hooke’s law. The derived engineering stresses and strains were synced and plotted to determine the effective compressvie modulus (E), yield point (σyield and εyield), and maximum strength (σmax 11

and εmax) of each osteon. E was defined as the slope of the stress-strain curve within the linear region using a best-fit algorithm which sequentially increased the data included in the regression [24]; in detail, the R2 value of the best fit line increases as data within the linear region is included in the regression, but then decreases with the addition of data from the non-linear region. As such, the point at which a maximum R2 value was reached was used to represent the yield point (σyield and εyield). Additionally, a damage onset strain (εDamageOnset) and the Degree of Whitening were identified from thresholded image stacks of the video data using an automated whitening detection algorithm (see Fig. S.5). εDamageOnset was identified as the strain associated with the first video frame at which it was possible to detect an increase in the degree of whitening. Degree of Whitening was determined from the change in whitening area between damage onset and the peak and is reported as a percent of the total in-plane area. Lastly, the elastic work (WElastic) and post-yield work (WPostyield)

were calculated for each osteon using trapezoidal numerical integration of the force-

deflection data in Matlab 2016b (Mathworks, Inc., Natick, Massachusetts, United States). WElastic was the area under the force-displacement curve within the elastic region and WPostyield

was the area under the curve between the yield point and the displacement resulting in

a 10% strain.

2.5 Statistics Shapiro-Wilk normality tests and Bartlett’s tests of homogeneity of variances were used to evaluate the normality of the structural, compositional, and mechanical properties of the osteons from either donor and to determine if the groups shared equal variances. Normally distributed properties with equal variance were evaluated using a two sample t-test to determine if there were any significant differences between the donors while normally distributed properties with unequal variance were evaluated using a Welch two sample ttest. Non-normally distributed properties with equal and unequal variance were evaluated using a Mann–Whitney U test to check for significant differences in osteon properties between donors. The threshold for statistical significance was set to p = 0.05. A subset of the osteons from both donors was isolated based on their morphology and a repeat 12

statistical analysis was performed to determine if inconsistencies in the internal structure of the osteons influenced any observed significant differences between donors. Spearman’s rank order-correlations were calculated to assess the relationships between the structural (BV/TV and Collagen Angle), compositional (TMD), and mechanical properties of each donor. Linear regression analyses were conducted on the relationships which exhibited moderate to strong (r > |0.6|) correlations.

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Results To assess morphology of the osteons, µCT data were visually evaluated. Osteons from

both sets were divided into three categories: intact osteons (Fig. 3a) with a vertical central Haversian canal; osteons with branching of the Haversian canal (Fig. 3b); or osteons with the Haversian canal inclined to the periphery of the osteon (Fig. 3c). The 93-year-old donor had six intact, two branching, and five inclined canal osteons while the 64-year-old donor had nine, two, and three, respectively. Three failure modes were identified based on visual damage accumulation within the osteons (Fig. 4): shear, uniform or bulging. Shear failure occurred when a single slip plane appeared during testing prior to failure (Fig. 4a). Uniform failure occurred when global widening and whitening of the osteon was observed (Fig. 4b). Bulging failure occurred when only localized deformation and whitening were observed at either the top or bottom of the osteon (Fig. 4c). No single predominant type of failure was observed for either donor. However, the osteons from the 94-year-old donor predominantly failed in either uniform or bulging modes, whereas the observed failure modes for the osteons from the 64-year-old donor were more evenly distributed (Fig. 5). Osteons with branching and Haversian canal inclination exhibited all three modes of failure, whereas intact osteons failed either uniformly or in bulging (Fig. 5). When assessing the intact osteons only, the ratio of uniform to bulging failure was higher (5:1) for the 94-year-old-donor than in the 64-year-old donor (3:4). Stress-strain plots are given in Fig. 6 and the determined structural, compositional, and mechanical properties of the osteons from both donors are summarized in Table 1. No 13

significant differences were found in D, CSA, BV/TV, or Collagen Angle of the extracted osteons from either donor. The H of the osteons from the younger donor (Mdn: 462 µm) was significantly higher (U=17.5, p<0.01) than the H of the osteons from the older donor (Mdn: 408 µm). The range in mean Collagen Angle (62 to 74°) indicated that the osteons present in both donors were predominantly dark, i.e. longitudinal, or grey, i.e. alternating. TMD (1214 ± 32 mg HA/cm3) and E (Mdn: 6.4 GPa) of the osteons from the older donor were significantly higher (U= 36, p= 0.029 and p= 0.037, respectively) than TMD (1186 ± 30 mg HA/cm3) and E (Mdn: 5.3 GPa) of the osteons from the younger donor. σmax for the osteons of the older donor (124.6 ± 16 MPa) was also found to be significantly higher (p=0.01) than that of the osteons from the younger donor (96.3 ± 33 MPa). No significant differences were found in σyield, ɛyield, ɛmax or ɛDamageOnset of the extracted osteons from either donor. ɛDamageOnset, the onset of whitening, occurred at strains over double that of the yield point for all osteons (Table 1). Degree of Whitening was significantly larger (p<0.01) in the older donor (76 ± 12 %) than in the younger donor (61 ± 14 %) (Fig. S.5). Degree of Whitening was found to be sensitive to failure mode; nearly 100% of the total, visible osteon surface area (Aw) exhibited whitening at 10% strain in the uniform failure osteons while Aw only reached a maximum of 50% in the shear and bulging failure osteons (Fig. 7). No significant differences were found in WElastic or WPost-yield of the osteons from either donor. When considering the intact osteons, only H and the Degree of Whitening were significantly different between donors (p=0.02 and p=0.02, respectively). H of the younger donor intact osteons (447 ± 38 µm) remained higher than that of the older donor intact osteons (403 ± 11 µm). The Degree of Whitening in the older donor intact osteons (82 ± 7.4 %) remained significantly higher than in the younger donor intact osteons (64 ± 15 %). No significant differences were found between the remaining structural, compositional or mechanical parameters of the intact osteons from either donor.

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Significant negative correlations were found between E (R2 = 0.43, p = 0.015), σmax (R2 = 0.54, p = 0.004), and WPost-yield (R2 = 0.72, p < 0.001) and Collagen Angle in the osteons from the 94-year-old donor; no significant correlations with Collagen Angle were found for the mechanical properties of the 63-year-old donor (Fig. 8). Significant positive correlations were found between E (R2 = 0.52, p = 0.003), σmax (R2 = 0.75, p << 0.001), and WPost-yield (R2 = 0.57, p = 0.002) and BV/TV in the osteons from the 63-year-old donor; no significant correlations with BV/TV were found for the mechanical properties of the 94-year-old donor (Fig. 8). No significant correlations with TMD were found in either donor.

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Discussion The aim of the present study was to establish a robust protocol for reproducing and

validating the compression testing experiments conducted on individual osteons reported by Ascenzi and Bonucci [11]. An experimental protocol was established such that osteons approximately 250 µm in diameter were successfully extracted from slices of cortical human bone and loaded in compression. Further, both µCT and SHG microscopy imaging methods were incorporated into the protocol to improve the structural and compositional assessment of each osteon. The evaluated structural properties of the individual osteons indicated a high degree of reproducibility in the extraction procedure. The standard deviation in D was less than 9% for both donors. Although there was a significant difference in H between donors, this was a result of the initial cortical bone slice thickness and not a result of the milling procedure. Moreover, the H to D ratio for the current study ranged from 1.6 – 1.8 to 1 nearly matching the range reported by Ascenzi and Bonnucci (1.8 – 2 to 1). Despite the use of two donors, the current study did not have a wide range in Collagen Angle, making it impossible to classify the osteons into light, grey, and dark groups. The limited range in Collagen Angle may also have been a result of the approach used to approximate the 3D orientation of the collagen fibers, since the samples were measured at a 15

single analyzer position. However, the locations of transverse vs. longitudinal fibrils can be easily discerned using this approach (Figure 2) as well as individually measured (Figure S.4). Distinct segments of longitudinal ([70]) and alternating ([40/70]) laminate layups were observed within the osteons from both donors (representative osteons shown in Figure S.4). Overall, the SHG results indicate the majority of the extracted osteons had more longitudinally aligned collagen fibers (i.e. “dark” or “grey” osteons), in agreement with [25,26]. In these larger studies on whole cross-sections of the femur diaphysis (n=67 and 37, respectively), the prevalence of longitudinal compared to transverse collagen fiber alignment was attributed to the complexity of the loading environment at the mid-diaphysis (i.e. distributed bending and prominent torsion). Several studies investigating the link between the local mechanical loading environment and collagen fibril alignment have shown weak, but significant trends for a greater proportion of longitudinal collagen fiber alignment in the anterolateral/lateral cortex and a greater proportion of transverse collagen fiber alignment in the posteromedial/medial cortex of the human femur mid-diaphysis [25–28]. This was linked to differences in the local mechanical loading history. Specifically, predominant longitudinal fiber alignment was found in regions under tension while predominant transverse fiber alignment was found in regions under compressive loading. However, the overarching conclusion in each of these studies is that high degree of intra-subject variability prohibits population wide patterning of collagen fiber orientation across the mid-diaphysis. In future studies, the use of x-ray diffraction (XRD) or small-angle x-ray scattering (SAXS) could be used to confirm the SHG results both in- and out-of-plane. An initial SHG scan of the entire cross-section could also be used to locate specific osteons for extraction prior to milling. When combined with the use of a spatial grid like that of Goldman et al. (2003, 2005), the measured material and mechanical properties in the target osteons could be stratified by their mechanical loading histories (anterolateral/posteromedial) [25–27] as well as by regions experiencing greater bone turnover (endosteal/periosteal) [25,29].

16

Although Haversian canal size has been reported to increase with age and disease [7], BV/TV was not significantly different between the donors in this study despite their nearly 30year age gap. This also may have been affected by the osteon selection criteria for circularity; however, the nature of the milling procedure prohibited the extraction of more elliptical osteons without inducing significant disruptions in the concentric lamellae. Although significant, there was only a 2% difference in mineralization (i.e. average TMD) between the osteons of either donor, with TMD of the older donor being higher than that of the younger donor. The bulk of bone tissue mineralization (~70%) occurs shortly after formation and then continues through slow enlargement of the carbon-substituted hydroxyapatite mineral nanocrystals over time [30,31]. These combined indicate that the harvested osteons were similar in “age”, meaning that the time between production and extraction of the osteons was similar for both donors. Further investigation using higher resolution µCT or quantitative backscattered election imaging (qBEI) could reveal more subtle difference in osteon maturity, including any spatial heterogeneity within individual osteons not visible at a 3 µm voxel resolution. In the present study, E of individual osteons ranged from 2.2 GPa to 11.3 GPa, which is consistent with the values reported by Ascenzi and Bonucci [11]. However, these values remain low compared to those derived from nanoindentation (17 to 27 GPa [17–19]) and micropillar compression (~30 GPa [20,21]) tests, conducted using either a single or cyclic loading-unloading cycle(s). In these experiments, the apparent moduli were derived from the unloading portion of the force-deflection curve, ensuring that any inelastic deformation incurred during the loading cycle did not affect the measured elastic properties. Future experiments conducted on individual osteons should consider incorporating a more complex loading curve to determine the degree of modulus underestimation caused by damage incurred during the loading phase. Although inelastic deformation may be contributing the low E values, a 900% recovery in stiffness would be required to reach expected values for the softest osteons within this study. 17

A recovery of this magnitude is highly unlikely. This brings into question the exact role of individual osteons within cortical bone. Previous studies have shown that the cement lines separating osteons from the more mineralized interstitial bone serve as a means of arresting crack propagation, increasing tissue-level toughness [5,32]. A systemic reduced stiffness in osteons compared to surrounding tissue would give rise to an even greater proclivity for diverting and arresting crack propagating. Using the methods presented in the study, cylindrical interstitial bone samples and neighboring osteons could be evaluated to probe this apparent tradeoff between maintaining tissue-level toughness via osteon formation and stiffness via mature mineralization in interstitial lamellae. Ascenzi and Bonucci found that osteons with primarily longitudinal collagen fiber alignment were able to support greater stresses in tension [10] and torsion [14], whereas osteons with alternating collagen fiber alignment in adjacent lamellae were able to support greater stresses in compression [11]. Although the current study included predominantly longitudinal or alternating osteons, the osteons with lower mean Collagen Angle had higher values of σmax under compressive loading, in agreement with the previous studies (Fig. 8). In the sample set retrieved from the younger donor, there were osteons that experienced stresses of over 100 MPa in addition to weaker osteons which failed at stresses as low as 20 MPa (Fig. 6). This wide range of σmax was not observed in the osteons of the older donor, where no osteon yielded below 90 MPa or failed below 100 MPa. E and σmax of the older donor osteons were significantly higher than those of the younger donor; this was paired with an elevated TMD in the older donor osteons. Mineralization has long been associated with increased stiffness in bone so it may explain the higher mechanical properties in the older donor osteons. The range in properties found in the younger donor osteons may be related to the greater variation in osteon morphometry found in cortical bone from younger donors [7,31]. Parameters such as diameter, wall thickness, and lacunar number per osteon in bone from younger donors have higher standard deviations than those from older donors, consistent with the findings of the present study. 18

Although yield and failure strains were not found to be statistically different between donors, 20% (n=3) of the osteons of the younger donor failed at a strain over twice that of the highest εmax of the osteons from the older donor. Similarly, the coefficient of variation for E, σmax, σyield, εyield, εDamageOnset and εmax was always higher for the younger donor than for the older donor, indicating more mechanical heterogeneity. The presence of both stiff and compliant osteons in the younger bone may indicate a better preparedness for the formation of microdamage. The presence of more compliant osteons may explain the tendency for young bone to develop diffuse damage over linear microcracks in response to cyclic loading [6]. In addition to parameters retrieved from the mechanical tests, videography data was evaluated to obtain insight into the failure mode of individual osteons during compression testing. Single plane shear failure occurred at a roughly 45° angle to the longitudinal axis of the osteons. In these cases, the internal shear forces exceeded the shear strength of the osteon, resulting in a fracture which propagated radially through the lamellae. This type of fracture is considered typical for ductile materials and has also been reported to occur under compression of both dry and hydrated bone micropillars harvested from osteonal lamellae [20,21]. In the case of uniform failure, damage accumulated globally and cracks appeared to propagate along the principle stress axis between the layers of lamellae throughout the whole length of the osteon; this resulted in a uniform distribution of whitening in the recorded video image (Fig. 7). The nature of this quasi-brittle failure mode may be due to the material and structural heterogeneity of the osteon (i.e. high mineralization at the cement lines, varying lamellar thickness, etc.). This same quasi-brittle behavior applies to the failure of cortical bone at a macro-level where fracture propagates between the lower structural components of bone, such as osteons and interstitial lamellae, in an effort to increase fracture toughness [6]. Notably, the majority of the uniform failure osteons were classified as intact (Fig. 5). As such, internal flaws like an inclined or branched Haversian canal precluded 19

a uniform failure by acting as stress concentration points, weakening the integrity of the osteon. In the case of bulging failure, accumulation of damage was confined to either the top or bottom surface of the compression cylinder, constraining crack propagation to a limited area. This could partly be explained by the high frictional forces occurring between the osteon and the compression cylinder during testing, leading to a redistribution of forces and accumulation of higher stresses at the corresponding end of the osteon. Additionally, bulging failure could indicate the presence of pre-existing flaws either contained in the osteon physiologically or induced during the sample preparation process. Regardless, the locally induced stresses led to the local accumulation of damage. Due to the high number of parameters that could have influenced the mechanical behavior of the osteons, this type of failure was regarded as a structural instability, rather than a true failure mode. It should be noted that the present classifications were made in accordance to the video taken from one side of the osteons; this may have resulted in a misinterpretation of the observed failure mode. For example, an osteon failing in bulging, if observed from another side, could have in fact failed in shear. The use of multiple cameras in future studies could mitigate this issue. Disregarding bulging, nearly all of the osteons of the older donor failed uniformly in a quasi-brittle manner, whereas osteons of the younger donor exhibited an even distribution of shear and uniform failure (Fig. 5). Despite the limited number of donors, the observation that older donor osteons fail in a more brittle manner than younger donor osteons agrees well with the consensus in the literature that human bone becomes more brittle with age. The onset of whitening, which is indicative of local damage formation [33–35], was observed in all osteons at strains near or, in some cases, after ɛmax. Previous studies conducted on larger bone samples, however, have reported that whitening is usually observed at the yield point [36,37]. This suggests that the whitening observed in large bone failure studies is indicative of failure within lower level ultrastructural components.

20

The linear regression analyses revealed an interesting dependency of the mechanical properties of the older donor osteons on Collagen Angle that was not present in the younger donor osteons. Moreover, the mechanical properties of the younger donor osteons appeared to be heavily dependent on the BV/TV, while the older donor osteons did not seem sensitive to Haversian canal size. This suggests that the stiffness and strength of osteons from the younger donor is a measure of the gross structural stability while these properties are dominated by the tissue level structural organization in the older donor osteons. These findings are, however, preliminary and require a larger sample and donor set to be fully validated. Despite the limited number of samples, the proposed method enables the quantification of inter-subject variability in structural, material, and mechanical properties, providing a platform for larger studies to investigate population differences between sexes, across age groups, or even between healthy and disease states.

5

Conclusion Individual osteons were extracted from human cortical bone with a high degree of

reproducibility and were robustly assessed. The osteons extracted from the 93-year-old donor were stiffer, more highly mineralized, and exhibited a strong dependence on the collagen orientation. In contrast, the osteons extracted from the 64-year-old donor exhibited more heterogeneity in the measured mechanical properties, which were heavily dependent on the gross structure of the osteon. The addition of more advanced imaging methods enabled the identification of distinct failure modes and confirmed that whitening observed during yielding of macroscale bone specimens represents failure at the local level.

Acknowledgments This work was supported by the Whitaker International Program. The funding source was not involved in the study design, in the data collection, analysis or interpretation, or in the decision to submit this article for publication.

Disclosures The authors declare that there is no conflict of interest. 21

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24

Figure Labels: Figure 1. Schematic showing the structural properties of the osteons determined using µCT. The Ferret diameter (D) was measured for each µCT slice and then averaged. The osteon height (H) was manually measured three times and then averaged. The cross-sectional area (CSA) was measured as the average annulus defined by the applied 110 grey value threshold through the height of the osteon, thus excluding the area of the Haversian canal. The bone volume fraction (BV/TV) was calculated from a cylindrical region of interest with outer diameter D and height H that included the Haversian canal. As a result, low BV/TV values were representative of a large Haversian canal size. The tissue mineral density (TMD) was determined using a density calibration equation to convert the mean grey value of the frontal osteonal cross-section to a physical density in mg HA/cm3. Figure 2. (Left) Schematic diagram of the second harmonic generation (SHG) microscopy technique used to approximate the collagen fibril orientation. (Center) Axial SHG microscopy images of characteristically dark (top) and light (bottom) osteons and (Right) projections of the respective collagen fiber orientation on the sagittal plane of representative osteons. Figure 3. µCT slices depicting an (a) intact osteon, (b) osteon with branching, and (c) osteon with Haversian canal inclined to its periphery. Osteons were scanned with a voxel resolution of 3.3µm at 70 kV and 145 mA. Figure 4. Graphical representation of the observed osteon failure modes: (a) single plane shear failure, (b) uniform compression failure, and (c) bulging, which occurred at either end of the osteon. Figure 5: (Left) Distribution of the observed failure modes for osteons from the 93-year-old (top) and 64-year-old (bottom) donors. (Right) Morphological classifications of the Haversian canal for thee osteons from each donor which failed in bulging, uniform, or shear failure modes. Figure 6: (Top) Stress-strain curves of all the tested osteons from the 64-year-old (Left) and 93-year-old (Right) donors with uniform (black), bulging (dashed), and shear (grey) failure modes labelled. (Bottom) Only the stress-strain curves from the osteons with intact morphology from either donor. Figure 6: (Top) Stress-strain curves of all the tested osteons from the 64-year-old (Left) and 93-year-old (Right) donors with uniform (black), bulging (dashed), and shear (grey) failure modes labelled. (Bottom) Only the stress-strain curves from the osteons with intact morphology from either donor. Figure 7: Outlined whitened area at selected time points for osteons from the 64 year-old donor, according to the stress vs strain and Degree of Whitening vs strain diagram on the right. Labeled time points are (0) initial strain, (1) εyield / 2, (2) εyield, (3) εDamageOnset, (4) σmax, (5) maximum Degree of Whitening / 2, (6) (10% strain + εDamageOnset) / 2, (7) 10% strain. (Top) Uniform failure mode showing a homogenous whitening area that covers almost the whole sample at 10% strain. (Middle) Bulging failure mode where whitening is restricted to the lower half of the sample. (Bottom) Shear failure mode where whitening mostly forms along a shear plane (indicated in black). Figure 8: E, σmax, and WPost-yield showed a significant correlation with Collagen Angle (Top) in the 93-year-old donor (black), while E, σmax, and WPost-yield showed a significant correlation with BV/TV (Bottom) in the 64-year-old donor.

25

Tables: Table 1: Summary of the mean and standard deviation of the structural, compositional, and mechanical parameters assessed for all of the individual osteons from the 64- and 93-yearold donors. Bolded values indicate significant differences between the two donors (p < 0.05). Donor Structural Property D (µm)* H (µm)* CSA (mm2) BV/TV ̅ (grey value)* (grey value)* Collagen Angle (°)** Composition Property TMD (mg/cm3) Mechanical Property E (GPa)* σyield (MPa) σmax (MPa) ɛyield ɛDamageOnset ɛmax* Degree of Whitening (%) WElastic (µJ) WPost-yield (µJ)

Age, 64

Age, 93

p

257 ± 22 462 ± 24 0.045 ± 0.006 0.88 ± 0.06 19.3 ± 5.2 177 ± 2.0 70 ± 8.9

255 ± 11 408 ± 20 0.046 ± 0.004 0.90 ± 0.04 23.3 ± 9.2 177 ± 13 68 ± 8.7

0.94 <0.01 0.48 0.29 0.52 0.66 0.55

1186 ± 31

1214 ± 33

0.037

5.3 ± 2.2 67.7 ± 23 96.3 ± 33 0.014 ± 0.004 0.025 ± 0.007 0.030 ± 0.048

6.4 ± 3.1 80.5 ± 16 125 ± 16 0.013 ± 0.004 0.028 ± 0.007 0.032 ± 0.008

0.029 0.11 0.010 0.69 0.39 0.79

61 ± 14

76 ± 12

<0.01

10.1 ± 5.3 165 ± 67

9.5 ± 3.7 172 ± 35

0.75 0.74

*Indicates when median values with IQ ranges are reported **Indicates when a composite mean and standard deviation are reported

26

Graphical Abstract

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