Is photobleaching necessary for Raman imaging of bone tissue using a green laser?

AbstractRaman microspectroscopy is widely used for musculoskeletal tissues studies. But the fluorescence background obscures prominent Raman bands of mineral and matrix components of bone tissue. A 532-nm laser irradiation has been used efficiently to remove the fluorescence background from Raman spectra of cortical bone. Photochemical bleaching reduces over 80% of the fluorescence background after 2 h and is found to be nondestructive within 40 min. The use of electron multiplying couple charge detector (EMCCD) enables to acquire Raman spectra of bone tissues within 1–5 s range and to obtain Raman images less than in 10 min

Investigating the Effects of Age and Exercise on Bone Composition and the Impact of Composition on Mechanical Integrity.

Fractures are the most frequent health problem associated with bone and represent a significant clinical and economic burden. Clinically, fracture risk is diagnosed by low bone mass and interventions to reduce fracture risk are designed to increase mass. However, aging and interventions, like exercise, influence fracture risk by more than what changes in mass predict, indicating that exercise and aging alter skeletal integrity by altering tissue quality, not just quantity. Currently, there is no clear understanding of how tissue quality contributes to skeletal integrity or how it can be altered by external influences. Therefore, this study examined the hypothesis that exercise and aging in adult mice would alter bone composition leading to altered mechanical competence, even when adjusting for changes to bone size and shape. Exercise significantly improved strength and resistance to fatigue-induced damage in young mice, but had no measured benefit in old mice. In young mice, exercise also increased mineralization and decreased carbonate substitution. Aging significantly reduced structural and tissue-level mechanical properties and increased mineral crystal size, carbonate substitution, and microcracking. Compositional changes with exercise and aging occurred in pre-existing bone (determined by micro-CT and calcein labeling) and mechanical improvements were observed without significant increases in bone size, demonstrating that bone can adapt to external stimuli by altering tissue quality without requiring modeling or remodeling. Further, colocalization of compositional and mechanical measurements by Raman microspectroscopy and nanoindentation provided corroborative evidence that the observed compositional changes contributed significantly to the observed changes in mechanical competence, but in an age dependent manner. This work challenges conventional theories about bone adaptation and the influence of bone composition on mechanical integrity. It was demonstrated for the first time that exercise and aging can modulate bone composition, and therefore tissue-level mechanical properties, even in the absence of bone formation or remodeling. Therefore, changes in tissue quality may often be overlooked because they may occur without significant changes in bone mass. This work also illustrates the potential utility of using compositional markers in diagnosing skeletal fragility but warns against making sweeping conclusions about the consequences of compositional changes in bone.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/62281/1/nsahar_1.pd

Exercise Alters Mineral and Matrix Composition in the Absence of Adding New Bone

The mechanical properties of bone are dictated by its amount, distribution and ‘quality’. The composition of the mineral and matrix phases is integral to defining ‘bone quality’. Exercise can potentially increase resistance to fracture, yet the effects of exercise on skeletal fragility, and how alterations in fragility are modulated by the amount, distribution and composition of bone, are unknown. In this investigation, the effects of exercise on the size, composition, mechanical properties and damage resistance of bones from mice of various ages, background strains and genetic makeup were assessed, as a means of testing the hypothesis that mechanical loading can improve skeletal fragility via compositional alterations. C57BL/6 mice (4-month-old males) ran on a treadmill for 21 days. Tibiae from exercised and control mice were analyzed for cross-sectional geometry, mechanical properties, microdamage and composition. Exercise significantly increased strength without increasing cross-sectional properties, suggesting that mechanical stimulation led to changes in the bone matrix, and these changes led to the improvements in mechanical properties. Consistent with this interpretation, the mineral/matrix ratio was significantly increased in exercised bones. The number of fatigue-induced microcracks was significantly lower in exercised bones, providing evidence that exercise modulates fatigue resistance. The ratio of nonreducible/reducible cross-links mirrored the damage data. Similar trends (exercise induced increases in mechanical properties without increases in cross-sectional properties, but with compositional changes) were also observed in 2-month-old biglycan-deficient and wild-type mice bred on a C57BL/6x129 genetic background

The bone diagnostic instrument III: Testing mouse femora

Here we describe modifications that allow the bone diagnostic instrument (BDI) [P. Hansma et al., Rev. Sci. Instrum. 79, 064303 (2008); Rev. Sci. Instrum. 77, 075105 (2006)], developed to test human bone, to test the femora of mice. These modifications include reducing the effective weight of the instrument on the bone, designing and fabricating new probe assemblies to minimize damage to the small bone, developing new testing protocols that involve smaller testing forces, and fabricating a jig for securing the smaller bones for testing. With these modifications, the BDI was used to test the hypothesis that short-term running has greater benefit on the mechanical properties of the femur for young growing mice compared to older, skeletally mature mice. We measured elastic modulus, hardness, and indentation distance increase (IDI), which had previously been shown to be the best discriminators in model systems known to exhibit differences in mechanical properties at the whole bone level. In the young exercised murine femora, the IDI was significantly lower than in young control femora. Since IDI has a relation to postyield properties, these results suggest that exercise during bone development increases post yield mechanical competence. We were also able to measure effects of aging on bone properties with the BDI. There was a significant increase in the IDI, and a significant decrease in the elastic modulus and hardness between the young and old groups. Thus, with the modifications described here, the BDI can take measurements on mouse bones and obtain statistically significant results

Calcium- and Phosphorus-Supplemented Diet Increases Bone Mass after Short-Term Exercise and Increases Bone Mass and Structural Strength after Long-Term Exercise in Adult Mice.

Exercise has long-lasting benefits to bone health that may help prevent fractures by increasing bone mass, bone strength, and tissue quality. Long-term exercise of 6-12 weeks in rodents increases bone mass and bone strength. However, in growing mice, a short-term exercise program of 3 weeks can limit increases in bone mass and structural strength, compared to non-exercised controls. Short-term exercise can, however, increase tissue strength, suggesting that exercise may create competition for minerals that favors initially improving tissue-level properties over structural-level properties. It was therefore hypothesized that adding calcium and phosphorus supplements to the diet may prevent decreases in bone mass and structural strength during a short-term exercise program, while leading to greater bone mass and structural strength than exercise alone after a long-term exercise program. A short-term exercise experiment was done for 3 weeks, and a long-term exercise experiment was done for 8 weeks. For each experiment, male 16-week old C57BL/6 mice were assigned to 4 weight-matched groups-exercise and non-exercise groups fed a control or mineral-supplemented diet. Exercise consisted of treadmill running at 12 m/min, 30 min/day for 7 days/week. After 3 weeks, exercised mice fed the supplemented diet had significantly increased tibial tissue mineral content (TMC) and cross-sectional area over exercised mice fed the control diet. After 8 weeks, tibial TMC, cross-sectional area, yield force, and ultimate force were greater from the combined treatments than from either exercise or supplemented diet alone. Serum markers of bone formation (PINP) and resorption (CTX) were both decreased by exercise on day 2. In exercised mice, day 2 PINP was significantly positively correlated with day 2 serum Ca, a correlation that was weaker and negative in non-exercised mice. Increasing dietary mineral consumption during an exercise program increases bone mass after 3 weeks and increases structural strength after 8 weeks, making bones best able to resist fracture

Tibial tissue-level mechanical properties following 8 weeks of exercise x mineral-supplemented diet.

<p>Data shown as mean ± standard deviation. Exercise significantly increased yield strain and pre-yield toughness. The supplemented diet did not affect tissue-level mechanical properties. *Significant exercise effect (*p < 0.05, ***p < 0.001, Two-way ANOVA). Groups connected by a horizontal bar are significantly different from each other (p < 0.05, Tukey’s test). C–non-exercised mice fed the control diet. D–non-exercised mice fed the supplemented diet. CE–exercised mice fed the control diet. DE–exercised mice fed the supplemented.</p

Tibial structural-level mechanical properties after 8 weeks of exercise x mineral-supplemented diet.

<p>Data shown as mean ± standard deviation. Combining the supplemented diet with exercise led to significantly greater structural strength than exercise or diet alone. *Significant exercise effect (*p < 0.05, **p < 0.01, ***p < 0.001, Two-way ANOVA). ^#Significant diet effect (#p < 0.05, ###p < 0.001, Two-way ANOVA). Groups connected by a horizontal bar are significantly different from each other (p < 0.05, Tukey’s test). C–non-exercised mice fed the control diet. D–non-exercised mice fed the supplemented diet. CE–exercised mice fed the control diet. DE–exercised mice fed the supplemented.</p

Significant treatment effects of exercise, diet, and diet and exercise interaction (p < 0.05 Two-way ANOVA).

<p>Significant treatment effects of exercise, diet, and diet and exercise interaction (p < 0.05 Two-way ANOVA).</p

Tibial cortical bone mineralization and cross-sectional geometric properties following 8 weeks of exercise x mineral-supplemented diet.

<p>Data shown as mean ± standard deviation. The mineral-supplemented diet significantly increased tissue mineral content (TMC), cortical area and moment of inertia in exercised and non-exercised mice, and volumetric tissue mineral density (vTMD) in non-exercised mice. *Significant exercise effect (p < 0.05, Two-way ANOVA). ^#Significant diet effect (###p < 0.001, Two-way ANOVA). Groups connected by a bar are significantly different from each other (p < 0.05, Tukey’s test). C–non-exercised mice fed the control diet. D–non-exercised mice fed the supplemented diet. CE–exercised mice fed the control diet. DE–exercised mice fed the supplemented.</p