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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2022)

Bone tissue “stress-whitens” (changes from translucent to opaque) immediately prior to fracture, yet the underlying mechanisms governing this phenomenon are unclear. Bone is comprised of three major constituents: a mineral phase (mostly hydroxyapatite), an organic phase (mostly collagen type I), and water. Past research has demonstrated that stress-whitening is a property of the collagen component, rather than the mineral component of bone. The hypothesis of the thesis is that stress-whitening is caused by a mechanically-driven change in the microstructure of the collagen component of bone. Label-free Raman microspectroscopy was used to measure changes in the chemical components of demineralized bone before and after tension. We demonstrate that mechanically caused stress-whitening in equine bone is associated with changes in the Raman spectrum. More specifically, the results of the Raman spectral data demonstrate that there is an effect of mechanical loading on the protein structure of bone, specifically as demonstrated by changes in the amide III (1280 cm-1), CH2 (1467 cm-1) and amide I (1671 cm-1) peak intensities. Interpreting these peak changes as related to collagen hydrogen bonding and bone toughness is consistent with an increased crystallization of collagen under mechanical load, which Janet Clark hypothesized as a toughening mechanism of collagenous tissues. The results of this research augment the understanding of the structural mechanisms of bone mechanics and have the potential to supplement the understanding of bone diseases associated with changes in molecular mechanics and disruption of intermolecular hydrogen bonding, such as osteogenesis imperfecta.

    Cover page: Bone Stress Whitening Is a Fundamental Molecular Mechanism of Collagen I
    • Thesis
    • Peer Reviewed
    UC Davis Electronic Theses and Dissertations (2021)

    Stress-fractures of bone are similar to fatigue fractures in engineering materials as both are caused by microdamage accumulation under repetitive loading. However, stress-fractures have a biologic component, since bone tissue is capable of repairing damage and/or adding mass in response to damaging loading. The cellular damage repair processes cause transient porosities to develop in bone tissue, which temporarily reduce modulus and increase the damage accumulation rate if damaging loading is continued. Consequently, the stress fracture process is an interaction between bone’s mechanical response to high-levels of loading (damage accumulation), and the biological responses of repair and hypertrophy that change tissue modulus.

    This work focuses on stress-reactions, bone tissue changes that precede a stress-fracture, in athletes. Specifically, racehorse proximal sesamoid bone (PSB) fracture is used as a naturally occurring model of an osteochondral stress fracture. The PSBs are a pair of bones in the metacarpophalangeal (fetlock) joint of the distal forelimb and PSB fracture is one of the most common fatal musculoskeletal injuries associated with race training. In this work, morphologic tissue properties between PSB from racehorses with (Case) and without (Control) a unilateral biaxial PSB are compared. Both Case and Control horses were in race-training at the time of death and their training histories were known. The observed tissue properties are related to exercise. In addition, a compartment model of bone’s turnover cycle is introduced and used to explain the associations among morphologic variables and exercise. The collected morphologic data is used to solve for the model’s steady-state rate constants. Finally, the relationship between PSB kinematics and how PSB motion may impact PSB fracture risk is explored.

    Our primary finding was that horses in higher intensity training develop a subchondral stress-reaction (lesion). A subchondral bone lesion was consistently found in the abaxial aspect of medial PSB from both Case fractured and Case intact contralateral PSB. This lesion was not found in Control PSBs. The lesion was characterized by low bone volume fraction, low tissue mineral density, and higher microdamage compared to surrounding tissue. The bilaterally of this lesion in Case horses coupled with the observed tissue properties make the lesion consistent with stress-reactions in subchondral tissue that develop before a complete stress-fracture. Within the subchondral lesion, bone volume fraction was negatively associated with exercise intensity and microcrack areal density was positively associated with exercise intensity. These findings imply the lesion was more severe in horses with more intense training. Generally, Controls had a less intense training program compared to Cases. Additionally, the bone volume fraction was higher in the internal trabecular bone of Case proximal sesamoid bones compared to Controls. At this internal site, microdamage was not observed and bone volume fraction increased with exercise intensity. Steady-state model rate constants were determined based on the observed morphologic tissue properties at these two locations. Modeling results suggest that the different relationships between exercise and tissue properties within the subchondral and internal sites will occur if model rate constants depend on exercise intensity in a location-specific manner. We hypothesize that the different relationships are due to a strain difference between the two regions that causes a damage-repair response in the subchondral region and an adaptive-response in the internal region. In vitro axial limb loading indicated that the PSBs may experience an articular incongruity at high-speed gallop loads. Further, kinematic analysis indicated that the medial PSB experienced external rotation at high-speed gallop loads, which may cause the stress-reaction to form on the abaxial surface of the medial PSB.

    The presented work is clinically relevant because the identified abaxial subchondral site can be examined for a lesion in vivo to test whether a horse has or is developing a stress reaction that puts it at risk of fracture. Also, the lesion was found to develop 1- 8 months after an increase in exercise intensity, making this time-frame an important period for clinical examinations. Finally, the calibrated compartment model could be incorporated into a predictive dynamic simulation to predict the effects of exercise on PSB morphology and lesion development.

      Cover page: High-speed Exercise and Fetlock Kinematics Affect the Development of Stress-Reactions in Racehorse Proximal Sesamoid Bones
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2013)
      The stretched exponential function has many applications in modeling numerous types of experimental relaxation data. However, problems arise when using standard algorithms to fit this function: we have observed that different initializations result in distinct fitted parameters. To avoid this problem, we developed a novel algorithm for fitting the stretched exponential model to relaxation data. This method is advantageous both because it requires only a single adjustable parameter and because it does not require initialization in the solution space. We tested this method on simulated data and experimental stress-relaxation data from bone and cartilage and found favorable results compared to a commonly-used Quasi-Newton method. For the simulated data, strong correlations were found between the simulated and fitted parameters suggesting that this method can accurately determine stretched exponential parameters. When this method was tested on experimental data, high quality fits were observed for both bone and cartilage stress-relaxation data that were significantly better than those determined with the Quasi-Newton algorithm.
        Cover page: A Novel Method for Curvefitting the Stretched Exponential Function to Experimental Data.
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2023)
        Focal bone lesions are often found prior to clinically relevant stress-fractures. Lesions are characterized by low bone volume fraction, low mineral density, and high levels of microdamage and are hypothesized to develop when bone tissue cannot sufficiently respond to damaging loading. It is difficult to determine how exercise drives the formation of these lesions because bone responds to mechanical loading and repairs damage. In this study, we derive steady-state rate constants for a compartment model of bone turnover using morphometric data from fractured and non-fractured racehorse proximal sesamoid bones (PSBs) and relate rate constants to racing-speed exercise data. Fractured PSBs had a subchondral focus of bone turnover and microdamage typical of lesions that develop prior to fracture. We determined steady-state model rate constants at the lesion site and an internal region without microdamage using bone volume fraction, tissue mineral density, and microdamage area fraction measurements. The derived undamaged bone resorption rate, damage formation rate, and osteoid formation rate had significant robust regression relationships to exercise intensity (rate) variables, layup (time out of exercise), and exercise 2-10 months before death. However, the direction of these relationships varied between the damaged (lesion) and non-damaged regions, reflecting that the biological response to damaging-loading differs from the response to non-damaging loading.
          Cover page: Training drives turnover rates in racehorse proximal sesamoid bones
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2009)
          Transforming and insulin-like growth factors are important in regulating bone mass. Thus, one would anticipate correlations between matrix concentrations of growth factors and functional properties of bone. We therefore investigated the relationships of (1) TGF-β2 and (2) IGF-I matrix concentrations with the trabecular microstructure, stress distribution, and mechanical properties of tibial cancellous bone from six male human cadavers. Trabecular stress amplification (VMExp/σapp) and variability (VMCOV) were calculated using microcomputed tomography (μCT)-based finite element simulations. Bone volume fraction (BV/TV), surface/volume ratio (BS/BV), trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp), connectivity (Eu.N), and anisotropy (DA) were measured using 3-D morphometry. Bone stiffness and strength were measured by mechanical testing. Matrix concentrations of TGF-β2 and IGF-I were measured by ELISA. We found higher matrix concentrations of TGF-β2 were associated with higher Tb.Sp and VMExp/σapp for pooled data and within subjects. Similarly, a higher matrix concentration of IGF-I was associated with lower stiffness, strength, BV/TV and Tb.Th and with higher BS/BV, Tb.Sp, VMExp/σapp and VMCOV for pooled data and within subjects. IGF-I and Tb.N were negatively associated within subjects. It appears variations of the stress distribution in cancellous bone correlate with the variation of the concentrations of TGF-β2 and IGF-I in bone matrix: increased local matrix concentrations of growth factors are associated with poor biomechanical and architectural properties of tibial cancellous bone.
            Cover page: Cancellous Bone Properties and Matrix Content of TGF-β2 and IGF-I in Human Tibia: A Pilot Study
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2015)
            Traumatic injuries can have systemic consequences, as the early inflammatory response after trauma can lead to tissue destruction at sites not affected by the initial injury. This systemic catabolism may occur in the skeleton following traumatic injuries such as anterior cruciate ligament (ACL) rupture. However, bone loss following injury at distant,unrelated skeletal sites has not yet been established. In the current study, we utilized a mouse knee injury model to determine whether acute knee injury causes a mechanically significant trabecular bone loss at a distant, unrelated skeletal site (L5 vertebral body).Knee injury was noninvasively induced using either high-speed (HS; 500 mm/s) or lowspeed(LS; 1 mm/s) tibial compression overload. HS injury creates an ACL rupture by midsubstance tear, while LS injury creates an ACL rupture with an associated avulsion bone fracture. At 10 days post-injury, vertebral trabecular bone structure was quantified using high-resolution microcomputed tomography (lCT), and differences in mechanical properties were determined using finite element modeling (FEM) and compressive mechanical testing. We hypothesized that knee injury would initiate a loss of trabecular bone structure and strength at the L5 vertebral body. Consistent with our hypothesis, we found significant decreases in trabecular bone volume fraction (BV/TV) and trabecular number at the L5 vertebral body in LS injured mice compared to sham (8.8% and 5.0%, respectively), while HS injured mice exhibited a similar, but lower magnitude response (5.1% and 2.5%, respectively). Contrary to our hypothesis, this decrease intrabecular bone structure did not translate to a significant deficit in compressive stiffness or ultimate load of the full trabecular body assessed by mechanical testing or FEM. However,we were able to detect significant decreases in compressive stiffness in both HS and LS injured specimens when FE models were loaded directly through the trabecular bone region (9.9% and 8.1%, and 3, respectively). This finding may be particularly important for osteoporotic fracture risk, as damage within vertebral bodies has been shown to initiate within the trabecular bone compartment. Altogether, these data point to a systemic trabecular bone loss as a consequence of fracture or traumatic musculoskeletal injury, which may be an underlying mechanism contributing to increased risk of refracture following an initial injury. This finding may have consequences for treatment of acute musculoskeletal injuries and the prevention of future bone fragility.
              Cover page: Trabecular bone loss at a distant skeletal site following noninvasive knee injury in mice.
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2014)
              Bone "stress-whitens," becoming visibly white during mechanical loading, immediately prior to failure. Stress-whitening is known to make materials tougher by dissipating mechanical energy. A greater understanding of stress-whitening, both an optical and mechanical phenomenon, may help explain age-related increases in fracture risk that occur without changes in bone mineralization. In this work, we directly measure the optical properties of demineralized bone as a function of deformation and immersing fluid (with different hydrogen-bonding potentials, water, and ethanol). The change in refractive index of demineralized bone was linear: with deformation and not applied force. Changes in refractive index were likely due to pushing low-refractive-index fluid out of specimens and secondarily due to changes in the refractive index of the collagenous phase. Results were consistent with stress-whitening of demineralized bone previously observed. In ethanol, the refractive index values were lower and less sensitive to deformation compared with deionized water, corroborating the sensitivity to fluid hydration. Differences in refractive index were consistent with structural changes in the collagenous phase such as densification that may also occur under mechanical loading. Understanding bone quality, particularly stress-whitening investigated here, may lead to new therapeutic targets and noninvasive methods to assess bone quality.
                Cover page: Strain-induced optical changes in demineralized bone.
                • Article
                • Peer Reviewed
                UC Davis Previously Published Works (2022)
                Medial proximal sesamoid bones (PSBs) from Thoroughbred racehorses that did (Case) or did not (Control) experience unilateral biaxial PSB fracture were evaluated for bone volume fraction (BVF), apparent mineral density (AMD), tissue mineral density (TMD), and microdamage in Case fractured, Case contralateral limb intact, and Control bones. A majority of Case bones had a subchondral lesion with high microdamage density, and low BVF, AMD, and TMD. Lesion microdamage and densitometric measures were associated with training history by robust linear regression. Exercise intensity was negatively related to BVF (0.07 ≤ R2  ≤ 0.12) and positively related to microcrack areal density (0.21 ≤ R2  ≤ 0.29) in the lesion; however, in an undamaged site, the relationships were opposite in direction. Regardless of location, TMD decreased with event frequency for both Case and Control, suggesting increased bone remodeling with exercise. Measures of how often animals were removed from active training (layups) predicted a decrease in TMD, AMD, BVF, and microdamage at regions away from the lesion site. A steady-state compartment model was used to organize the differences in the correlations between variables within the data set. The overall conclusions are that at the osteopenic lesion site, repair of microdamage by remodeling was not successful (e.g., lower bone mass, increased damage, and lower mineralization) but that in regions away from the lesion remodeling successfully controlled damage (e.g., higher bone mass, less microdamage, and lower mineralization).
                  Cover page: Exercise history predicts focal differences in bone volume fraction, mineral density and microdamage in the proximal sesamoid bones of Thoroughbred racehorses
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2019)
                  The most reliable predictor of future fracture risk is a previous fracture of any kind. The etiology of this increased fracture risk is not fully known, but it is possible that fracture initiates systemic bone loss, leading to greater fracture risk at all skeletal sites. In this study, we investigated systemic bone loss and recovery after femoral fracture in young (3-month-old) and middle-aged (12-month-old) mice. Transverse femur fractures were created using a controlled impact, and whole-body bone mineral density (BMD), trabecular and cortical microstructure, bone mechanical properties, bone formation and resorption rates, mouse voluntary movement, and systemic inflammation were quantified at multiple time points post-fracture. We found that fracture led to decreased whole-body BMD in both young and middle-aged mice 2 weeks post-fracture; this bone loss was recovered by 6 weeks in young but not middle-aged mice. Similarly, trabecular bone volume fraction (BV/TV) of the L5 vertebral body was significantly reduced in fractured mice relative to control mice 2 weeks post-fracture (-11% for young mice, -18% for middle-aged mice); no significant differences were observed 6 weeks post-fracture. At 3 days post-fracture, we observed significant increases in serum levels of interleukin-6 and significant decreases in voluntary movement in fractured mice compared with control mice, with considerably greater changes in middle-aged mice than in young mice. At this time point, we also observed increased osteoclast number on L5 vertebral body trabecular bone of fractured mice compared with control mice. These data show that systemic bone loss occurs after fracture in both young and middle-aged mice, and recovery from this bone loss may vary with age. This systemic response could contribute to increased future fracture risk after fracture; these data may inform clinical treatment of fractures with respect to improving long-term skeletal health. © 2018 American Society for Bone and Mineral Research.
                    Cover page: Age Dependence of Systemic Bone Loss and Recovery Following Femur Fracture in Mice
                    • Article
                    • Peer Reviewed
                    UC Davis Previously Published Works (2014)
                    There is a need for improved biomaterials for use in treating non-healing bone defects. A number of natural and synthetic biomaterials have been used for the regeneration of bone tissue with mixed results. One approach is to modify native tissue via decellularization or other treatment for use as natural scaffolding for tissue repair. In this study, our goal was to improve on our previously published alternating solution immersion (ASI) method to fabricate a robust, biocompatible, and mechanically competent biomaterial from natural demineralized bone matrix (DBM). The improved method includes an antigen removal (AR) treatment step which improves mineralization and stiffness while removing unwanted proteins. The chemistry of the mineral in the remineralized bone matrix (RBM) was consistent with dicalcium phosphate dihydrate (brushite), a material used clinically in bone healing applications. Mass spectrometry identified proteins removed from the matrix with AR treatment to include α-2 HS-glycoprotein and osteopontin, noncollagenous proteins (NCPs) and known inhibitors of biomineralization. Additionally, the RBM supported the survival, proliferation, and differentiation of human mesenchymal stromal cells (MSCs) in vitro as well or better than other widely used biomaterials including DBM and PLG scaffolds. DNA content increased more than 10-fold on RBM compared to DBM and PLG; likewise, osteogenic gene expression was significantly increased after 1 and 2 weeks. We demonstrated that ASI remineralization has the capacity to fabricate mechanically stiff and biocompatible RBM, a suitable biomaterial for cell culture applications.
                      Cover page: Remineralized bone matrix as a scaffold for bone tissue engineering
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