Fourier Transform Infrared Spectroscopic Imaging of Fracture Healing in the Normal Mouse.

Fourier transform infrared spectroscopic imaging (FTIRI) was used to study bone healing with spatial analysis of various callus tissues in wild type mice. Femoral fractures were produced in 28 male C57BL mice by osteotomy. Animals were sacrificed at 1, 2, 4, and 8 weeks to obtain callus tissue at well-defined healing stages. Following microcomputerized tomography, bone samples were cut in consecutive sections for FTIRI and histology, allowing for spatial correlation of both imaging methods in different callus areas (early calcified cartilage, woven bone, areas of intramembranous and endochondral bone formation). Based on FTIRI, mineral/matrix ratio increased significantly during the first 4 weeks of fracture healing in all callus areas and correlated with bone mineral density measured by micro-CT. Carbonate/phosphate ratio was elevated in newly formed calcified tissue and at week 2 attained values comparable to cortical bone. Collagen maturity and mineral crystallinity increased during weeks 1-8 in most tissues while acid phosphate substitution decreased. Temporal and callus area dependent changes were detected throughout the healing period. These data assert the usefulness of FTIRI for evaluation of fracture healing in the mouse and its potential to evaluate pathologic fracture healing and the effects of therapeutic interventions.

Intermittent PTH administration and mechanical loading are anabolic for periprosthetic cancellous bone.

The purpose of this study was to determine the individual and combined effects on periprosthetic cancellous bone of intermittent parathyroid hormone administration (iPTH) and mechanical loading at the cellular, molecular, and tissue levels. Porous titanium implants were inserted bilaterally on the cancellous bone of adult rabbits beneath a loading device attached to the distal lateral femur. The left femur received a sham loading device. The right femur was loaded daily, and half of the rabbits received daily PTH. Periprosthetic bone was evaluated up to 28 days for gene expression, histology, and µCT analysis. Loading and iPTH increased bone mass by a combination of two mechanisms: (1) Altering cell populations in a pro-osteoblastic/anti-adipocytic direction, and (2) controlling bone turnover by modulating the RANKL-OPG ratio. At the tissue level, BV/TV increased with both loading (+53%, p < 0.05) and iPTH (+54%, p < 0.05). Combined treatment showed only small additional effects at the cellular and molecular levels that corresponded to a small additive effect on bone volume (+13% compared to iPTH alone, p > 0.05). This study suggests that iPTH and loading are potential therapies for enhancing periprosthetic bone formation. The elucidation of the cellular and molecular response may help further enhance the combined therapy and related targeted treatment strategies.

Skeletal response of male mice to anabolic hormone therapy in the absence of the Igfals gene.

IGF-I is a critical regulator of skeletal acquisition, which acts in endocrine and autocrine/paracrine modes. In serum, IGF-I is carried by the IGF-binding proteins in binary complexes. Further stabilization of these complexes is achieved by binding to the acid labile subunit (ALS) in a ternary complex (of IGF-I-IGF-binding protein 3/5-ALS). Ablation of the Igfals gene in humans (ALS deficiency) and mice (ALS knockout [ALSKO]) leads to markedly decreased serum IGF-I levels, growth retardation, and impaired skeletal acquisition. To investigate whether hormonal replacement therapy would improve the skeletal phenotype in cases of Igfals gene ablation, we treated male ALSKO mice with GH, IGF-I, or a combination of both. Treatments were administered to animals between 4 and 16 weeks of age or from 8 to 16 weeks of age. Although all treatment groups showed an increase (20%) in serum IGF-I levels, there was no increase in body weight, weight gain, or bone length in either age group. Despite the blunted linear growth in response to hormone therapy, ALSKO mice treated with GH showed radial bone growth, which contributed to bone strength tested by 4-point bending. We found that ALSKO mice treated with GH showed increased total cross-sectional area, cortical bone area, and cortical thickness by microtomography. Dynamic histomorphometry showed that although GH and double treatment groups resulted in trends towards increased bone formation parameters, these did not reach significance. However, bone resorption parameters were significantly increased in all treatment groups. ALSKO mice treated between 4 and 16 weeks of age showed minor differences in bone traits compared with vehicle-treated mice. In conclusion, treatment with GH and IGF-I do not work synergistically to rescue the stunted growth found in mice lacking the Igfals gene. Although GH alone appears to increase bone parameters slightly, it does not affect body weight or linear growth.

Low levels of plasma IGF-1 inhibit intracortical bone remodeling during aging.

Studies linking insulin-like growth factor-1 (IGF-1) to age-related bone loss in humans have been reported but remain only correlative. In this investigation, we characterized the bone phenotype of aged WT C57BL/6J male mice in comparison to that of C57BL/6J mice with reduced serum IGF-1 levels arising from an igfals gene deletion (ALS knockout (ALSKO)). During the aging process, WT mice showed an increase in fat mass and decrease lean mass while ALSKO mice had stable lean and fat mass values. Skeletal analyses of femora from WT mice revealed an expansion of the marrow area and a significant accumulation of intracortical porosity associated with increased intracortical remodeling. In contrast, ALSKO mice showed only small age-related declines in the amount of cortical bone tissue and minimal intracortical porosity, at 2 years of age. Accordingly, mechanical tests of femora from 2-year-old WT mice revealed reduced stiffness and maximal load when compared to bones from ALSKO mice. We show here that lifelong reductions in serum IGF-1 compromise skeletal size in development leading to slender bones; they are also associated with decreased intracortical bone remodeling and preservation of bone strength during aging.

Unbound (bioavailable) IGF1 enhances somatic growth.

Understanding insulin-like growth factor-1 (IGF1) biology is of particular importance because, apart from its role in mediating growth, it plays key roles in cellular transformation, organ regeneration, immune function, development of the musculoskeletal system and aging. IGF1 bioactivity is modulated by its binding to IGF-binding proteins (IGFBPs) and the acid labile subunit (ALS), which are present in serum and tissues. To determine whether IGF1 binding to IGFBPs is necessary to facilitate normal growth and development, we used a gene-targeting approach and generated two novel knock-in mouse models of mutated IGF1, in which the native Igf1 gene was replaced by Des-Igf1 (KID mice) or R3-Igf1 (KIR mice). The KID and KIR mutant proteins have reduced affinity for the IGFBPs, and therefore present as unbound IGF1, or 'free IGF1'. We found that both KID and KIR mice have reduced serum IGF1 levels and a concomitant increase in serum growth hormone levels. Ternary complex formation of IGF1 with the IGFBPs and the ALS was markedly reduced in sera from KID and KIR mice compared with wild type. Both mutant mice showed increased body weight, body and bone lengths, and relative lean mass. We found selective organomegaly of the spleen, kidneys and uterus, enhanced mammary gland complexity, and increased skeletal acquisition. The KID and KIR models show unequivocally that IGF1-complex formation with the IGFBPs is fundamental for establishing normal body and organ size, and that uncontrolled IGF bioactivity could lead to pathological conditions.

Increased serum IGF-1 levels protect the musculoskeletal system but are associated with elevated oxidative stress markers and increased mortality independent of tissue igf1 gene expression.

Although the literature suggests a protective (anabolic) effect of insulin-like growth factor-1 (IGF-1) on the musculoskeletal system during growth and aging, there is evidence that reductions in IGF-1 signaling are advantageous for promoting an increase in life span through reduction in oxidative stress-induced tissue damage. To better understand this paradox, we utilized the hepatocyte-specific IGF-1 transgenic (HIT) mice, which exhibit 3-fold increases in serum IGF-1, with normal IGF-1 expression in other tissues, and mice with an IGF-1 null background that exclusively express IGF-1 in the liver, which thereby deliver IGF-1 by the endocrine route only (KO-HIT mice). We found that in the total absence of tissue igf1 gene expression (KO-HIT), increases in serum IGF-1 levels were associated with increased levels of lipid peroxidation products in serum and increased mortality rate at 18 months of age in both genders. Surprisingly, however, we found that in female mice, tissue IGF-1 plays an important role in preserving trabecular bone architecture as KO-HIT mice show bone loss in the femoral distal metaphysis. Additionally, in male KO-HIT mice, increases in serum IGF-1 levels were insufficient to protect against age-related muscle loss.

Serum IGF-1 affects skeletal acquisition in a temporal and compartment-specific manner.

Insulin-like growth factor-1 (IGF-1) plays a critical role in the development of the growing skeleton by establishing both longitudinal and transverse bone accrual. IGF-1 has also been implicated in the maintenance of bone mass during late adulthood and aging, as decreases in serum IGF-1 levels appear to correlate with decreases in bone mineral density (BMD). Although informative, mouse models to date have been unable to separate the temporal effects of IGF-1 depletion on skeletal development. To address this problem, we performed a skeletal characterization of the inducible LID mouse (iLID), in which serum IGF-1 levels are depleted at selected ages. We found that depletion of serum IGF-1 in male iLID mice prior to adulthood (4 weeks) decreased trabecular bone architecture and significantly reduced transverse cortical bone properties (Ct.Ar, Ct.Th) by 16 weeks (adulthood). Likewise, depletion of serum IGF-1 in iLID males at 8 weeks of age, resulted in significantly reduced transverse cortical bone properties (Ct.Ar, Ct.Th) by 32 weeks (late adulthood), but had no effect on trabecular bone architecture. In contrast, depletion of serum IGF-1 after peak bone acquisition (at 16 weeks) resulted in enhancement of trabecular bone architecture, but no significant changes in cortical bone properties by 32 weeks as compared to controls. These results indicate that while serum IGF-1 is essential for bone accrual during the postnatal growth phase, depletion of IGF-1 after peak bone acquisition (16 weeks) is compartment-specific and does not have a detrimental effect on cortical bone mass in the older adult mouse.

Growth hormone mediates pubertal skeletal development independent of hepatic IGF-1 production.

Deficiencies in either growth hormone (GH) or insulin-like growth factor 1 (IGF-1) are associated with reductions in bone size during growth in humans and animal models. Liver-specific IGF-1-deficient (LID) mice, which have 75% reductions in serum IGF-1, were created previously to separate the effects of endocrine (serum) IGF-1 from autocrine/paracrine IGF-1. However, LID mice also have two- to threefold increases in GH, and this may contribute to the observed pubertal skeletal phenotype. To clarify the role of GH in skeletal development under conditions of significantly reduced serum IGF-1 levels (but normal tissue IGF-1 levels), we studied the skeletal response of male LID and control mice to GH inhibition by pegvisomant from 4 to 8 weeks of age. Treatment of LID mice with pegvisomant resulted in significant reductions in body weight, femur length (Le), and femur total area (Tt.Ar), as well as further reductions in serum IGF-1 levels by 8 weeks of age, compared with the mean values of vehicle-treated LID mice. Reductions in both Tt.Ar and Le were proportional after treatment with pegvisomant. On the other hand, the relative amount of cortical tissue formed (RCA) in LID mice treated with pegvisomant was significantly less than that in both vehicle-treated LID and control mice, indicating that antagonizing GH action, either directly (through GH receptor signaling inhibition) or indirectly (through further reductions in serum/tissue IGF-1 levels), results in disproportionate reductions in the amount of cortical bone formed. This resulted in bones with significantly reduced mechanical properties (femoral whole-bone stiffness and work to failure were markedly decreased), suggesting that compensatory increases of GH in states of IGF-1 deficiency (LID mice) act to protect against a severe inhibition of bone modeling during growth, which otherwise would result in bones that are too weak for normal and/or extreme loading conditions.

Measuring the dynamic mechanical response of hydrated mouse bone by nanoindentation.

This study demonstrates a novel approach to characterizing hydrated bone's viscoelastic behavior at lamellar length scales using dynamic indentation techniques. We studied the submicron-level viscoelastic response of bone tissue from two different inbred mouse strains, A/J and B6, with known differences in whole bone and tissue-level mechanical properties. Our results show that bone having a higher collagen content or a lower mineral-to-matrix ratio demonstrates a trend towards a larger viscoelastic response. When normalized for anatomical location relative to biological growth patterns in the antero-medial (AM) cortex, bone tissue from B6 femora, known to have a lower mineral-to-matrix ratio, is shown to exhibit a significantly higher viscoelastic response compared to A/J tissue. Newer bone regions with a higher collagen content (closer to the endosteal edge of the AM cortex) showed a trend towards a larger viscoelastic response. Our study demonstrates the feasibility of this technique for analyzing local composition-property relationships in bone. Further, this technique of viscoelastic nanoindentation mapping of the bone surface at these submicron length scales is shown to be highly advantageous in studying subsurface features, such as porosity, of wet hydrated biological specimens, which are difficult to identify using other methods.

IGF-1 and bone: New discoveries from mouse models.

Insulin-like growth factor-1 (IGF-1) plays a central role in cellular growth, differentiation, survival, and cell cycle progression. It is expressed early during development and its effects are mediated through binding to a tyrosine kinase receptor, the insulin-like growth factor-1 receptor (IGF-1R). In the circulation, the IGFs bind to IGF-binding proteins (IGFBPs), which determine their bioavailability and regulate the interaction between the IGFs and IGF-1R. Studies in animal models and in humans have established critical roles for IGFs in skeletal growth and development. In this review we present new and old findings from mouse models of the IGF system and discuss their clinical relevance to normal and pathological skeletal physiology.

Elevated serum IGF-1 levels synergize PTH action on the skeleton only when the tissue IGF-1 axis is intact.

There is growing evidence that insulin-like growth factor 1 (IGF-1) and parathyroid hormone (PTH) have synergistic actions on bone and that part of the anabolic effects of PTH is mediated by local production of IGF-1. In this study we analyzed the skeletal response to PTH in mouse models with manipulated endocrine or autocrine/paracrine IGF-1. We used mice carrying a hepatic IGF-1 transgene (HIT), which results in a threefold increase in serum IGF-1 levels and normal tissue IGF-1 expression, and Igf1 null mice with blunted IGF-1 expression in tissues but threefold increases in serum IGF-1 levels (KO-HIT). Evaluation of skeletal growth showed that elevations in serum IGF-1 in mice with Igf1 gene ablation in all tissues except the liver (KO-HIT) resulted in a restoration of skeletal morphology and mechanical properties by adulthood. Intermittent PTH treatment of adult HIT mice resulted in increases in serum osteocalcin levels, femoral total cross-sectional area, cortical bone area and cortical bone thickness, as well as bone mechanical properties. We found that the skeletal response of HIT mice to PTH was significantly higher than that of control mice, suggesting synergy between IGF-1 and PTH on bone. In sharp contrast, although PTH-treated KO-HIT mice demonstrated an anabolic response in cortical and trabecular bone compartments compared with vehicle-treated KO-HIT mice, their response was identical to that of PTH-treated control mice. We conclude that (1) in the presence of elevated serum IGF-1 levels, PTH can exert an anabolic response in bone even in the total absence of tissue IGF-1, and (2) elevations in serum IGF-1 levels synergize PTH action on bone only if the tissue IGF-1 axis is intact. Thus enhancement of PTH anabolic actions depends on tissue IGF-1.

Sex-specific regulation of body size and bone slenderness by the acid labile subunit.

Insulin-like growth factor 1 (IGF-1) is a crucial mediator of body size and bone mass during growth and development. In serum, IGF-1 is stabilized by several IGF-1-binding proteins (IGFBPs) and the acid labile subunit (ALS). Previous research using ALS knockout (ALSKO) mice indicated a growth retardation phenotype, and clinical reports of humans have indicated short stature and low bone mineral density (BMD) in patients with ALS deficiency. To determine the temporal and sex-specific effects of ALS deficiency on body size and skeletal development during growth, we characterized control and ALSKO mice from 4 to 16 weeks of age. We found that female ALSKO mice had an earlier-onset reduction in body size (4 weeks) but that both female and male ALSKO mice were consistently smaller than control mice. Interestingly, skeletal analyses at multiple ages showed increased slenderness of ALSKO femurs that was more severe in females than in males. Both male and female ALSKO mice appeared to compensate for their more slender bones through increased bone formation on their endosteal surfaces during growth, but ALSKO females had increased endosteal bone formation compared with ALSKO males. This study revealed age- and sex-specific dependencies of body size and bone size on the ALS. These findings may explain the heterogeneity in growth and BMD measurements reported in human ALS-deficient patients.

Elevated serum levels of IGF-1 are sufficient to establish normal body size and skeletal properties even in the absence of tissue IGF-1.

Use of recombinant insulin-like growth factor 1 (IGF-1) as a treatment for primary IGF-1 deficiency in children has become increasingly common. When untreated, primary IGF-1 deficiency may lead to a range of metabolic disorders, including lipid abnormalities, insulin resistance, and decreased bone density. To date, results of this therapy are considered encouraging; however, our understanding of the role played by IGF-1 during development remains limited. Studies on long-term treatment with recombinant IGF-1 in both children and animals are few. Here, we used two novel transgenic mouse strains to test the long-term effects of elevated circulating IGF-1 on body size and skeletal development. Overexpression of the rat igf1 transgene in livers of mice with otherwise normal IGF-1 expression (HIT mice) resulted in approximately threefold increases in serum IGF-1 levels throughout growth, as well as greater body mass and enhanced skeletal size, architecture, and mechanical properties. When the igf1 transgene was overexpressed in livers of igf1 null mice (KO-HIT), the comparably elevated serum IGF-1 failed to overcome growth and skeletal deficiencies during neonatal and early postnatal growth. However, between 4 and 16 weeks of age, increased serum IGF-1 fully compensated for the absence of locally produced IGF-1 because body weights and lengths of KO-HIT mice became comparable with controls. Furthermore, micro-computed tomography (microCT) analysis revealed that early deficits in skeletal structure of KO-HIT mice were restored to control levels by adulthood. Our data indicate that in the absence of tissue igf1 gene expression, maintaining long-term elevations in serum IGF-1 is sufficient to establish normal body size, body composition, and both skeletal architecture and mechanical function.

Genetically determined phenotype covariation networks control bone strength.

To identify genes affecting bone strength, we studied how genetic variants regulate components of a phenotypic covariation network that was previously shown to accurately characterize the compensatory trait interactions involved in functional adaptation during growth. Quantitative trait loci (QTLs) regulating femoral robustness, morphologic compensation, and mineralization (tissue quality) were mapped at three ages during growth using AXB/BXA Recombinant Inbred (RI) mouse strains and adult B6-i(A) Chromosome Substitution Strains (CSS). QTLs for robustness were identified on chromosomes 8, 12, 18, and 19 and confirmed at all three ages, indicating that genetic variants established robustness postnatally without further modification. A QTL for morphologic compensation, which was measured as the relationship between cortical area and body weight, was identified on chromosome 8. This QTL limited the amount of bone formed during growth and thus acted as a setpoint for diaphyseal bone mass. Additional QTLs were identified from the CSS analysis. QTLs for robustness and morphologic compensation regulated bone structure independently (ie, in a nonpleiotropic manner), indicating that each trait may be targeted separately to individualize treatments aiming to improve strength. Multiple regression analyses showed that variation in morphologic compensation and tissue quality, not bone size, determined femoral strength relative to body weight. Thus an individual inheriting slender bones will not necessarily inherit weak bones unless the individual also inherits a gene that impairs compensation. This systems genetic analysis showed that genetically determined phenotype covariation networks control bone strength, suggesting that incorporating functional adaptation into genetic analyses will advance our understanding of the genetic basis of bone strength.

Serum IGF-1 determines skeletal strength by regulating subperiosteal expansion and trait interactions.

Strong correlations between serum IGF-1 levels and fracture risk indicate that IGF-1 plays a critical role in regulating bone strength. However, the mechanism by which serum IGF-1 regulates bone structure and fracture resistance remains obscure and cannot be determined using conventional approaches. Previous analysis of adult liver-specific IGF-1-deficient (LID) mice, which exhibit 75% reductions in serum IGF-1 levels, showed reductions in periosteal circumference, femoral cross-sectional area, cortical thickness, and total volumetric BMD. Understanding the developmental sequences and the resultant anatomical changes that led to this adult phenotype is the key for understanding the complex relationship between serum IGF-1 levels and fracture risk. Here, we identified a unique developmental pattern of morphological and compositional traits that contribute to bone strength. We show that reduced bone strength associated with low levels of IGF-1 in serum (LID mice) result in impaired subperiosteal expansion combined with impaired endosteal apposition and lack of compensatory changes in mineralization throughout growth and aging. We show that serum IGF-1 affects cellular activity differently depending on the cortical surface. Last, we show that chronic reductions in serum IGF-1 indirectly affect bone strength through its effect on the marrow myeloid progenitor cell population. We conclude that serum IGF-1 not only regulates bone size, shape, and composition during ontogeny, but it plays a more fundamental role-that of regulating an individual's ability to adapt its bone structure to mechanical loads during growth and development.

Genetic variation in mouse femoral tissue-level mineral content underlies differences in whole bone mechanical properties.

A/J mice, as compared to C57BL/6J (B6) mice, have a significantly greater total femoral mineral (ash) content which correlates with an increased femoral stiffness (resistance to deformation), but also with an increased brittleness (catastrophic failure). To determine if this whole bone variation in mineral content is indicative of significant mineral and/or matrix variation at the tissue level, femora from 16-week-old female A/J and B6 mice were isolated, embedded in PMMA, sectioned and mounted on barium fluoride infrared windows for FTIRI analyses. In addition, preliminary studies of femora from C3H/HeJ (C3H) mice were conducted, since they have an ash content intermediate to A/J and B6. Mean values for mineral-to-matrix ratio were significantly different for A/J (8.4 +/- 0.8) and B6 (7.5 +/- 0.4), as were values for collagen cross-link maturity (1.8 +/- 0.05 and 3.2 +/- 0.1, respectively). C3H mice appeared to have a mineral-to-matrix ratio intermediate of A/J and B6, and cross-link maturity greater than both A/J and B6. B6 femora had similar carbonate-to-amide ratios, carbonate-to-mineral ratios and acid phosphate levels. Thus, whole bone differences in mineral content are concurrent with tissue-level variation in mineral content and collagen maturity in inbred mice. The greater stiffness and brittleness of A/J femora are likely due to differential biological control (osteoblast activity) of the amount of mineral.

Phenotypic integration of skeletal traits during growth buffers genetic variants affecting the slenderness of femora in inbred mouse strains.

Compensatory interactions among adult skeletal traits are critical for establishing strength but complicate the search for fracture susceptibility genes by allowing many genetic variants to exist in a population without loss of function. A better understanding of how these interactions arise during growth will provide new insight into genotype-phenotype relationships and the biological controls that establish skeletal strength. We tested the hypothesis that genetic variants affecting growth in width relative to growth in length (slenderness) are coordinated with movement of the inner bone surface and matrix mineralization to match stiffness with weight-bearing loads during postnatal growth. Midshaft femoral morphology and tissue-mineral density were quantified at ages of 1 day and at 4, 8, and 16 weeks for a panel of 20 female AXB/BXA recombinant inbred mouse strains. Path Analyses revealed significant compensatory interactions among outer-surface expansion rate, inner-surface expansion rate, and tissue-mineral density during postnatal growth, indicating that genetic variants affecting bone slenderness were buffered mechanically by the precise regulation of bone surface movements and matrix mineralization. Importantly, the covariation between morphology and mineralization resulted from a heritable constraint limiting the amount of tissue that could be used to construct a functional femur. The functional interactions during growth explained 56-99% of the variability in adult traits and mechanical properties. These functional interactions provide quantitative expectations of how genetic or environmental variants affecting one trait should be compensated by changes in other traits. Variants that impair this process or that cannot be fully compensated are expected to alter skeletal growth leading to underdesigned (weak) or overdesigned (bulky) structures.

Genetic architecture of complex traits: large phenotypic effects and pervasive epistasis.

The genetic architecture of complex traits underlying physiology and disease in most organisms remains elusive. We still know little about the number of genes that underlie these traits, the magnitude of their effects, or the extent to which they interact. Chromosome substitution strains (CSSs) enable statistically powerful studies based on testing engineered inbred strains that have single, unique, and nonoverlapping genetic differences, thereby providing measures of phenotypic effects that are attributable to individual chromosomes. Here, we report a study of phenotypic effects and gene interactions for 90 blood, bone, and metabolic traits in a mouse CSS panel and 54 traits in a rat CSS panel. Two key observations emerge about the genetic architecture of these traits. First, the traits tend to be highly polygenic: across the genome, many individual chromosome substitutions each had significant phenotypic effects and, within each of the chromosomes studied, multiple distinct loci were found. Second, strong epistasis was found among the individual chromosomes. Specifically, individual chromosome substitutions often conferred surprisingly large effects (often a substantial fraction of the entire phenotypic difference between the parental strains), with the result that the sum of these individual effects often dramatically exceeded the difference between the parental strains. We suggest that strong, pervasive epistasis may reflect the presence of several phenotypically-buffered physiological states. These results have implications for identification of complex trait genes, developmental and physiological studies of phenotypic variation, and opportunities to engineer phenotypic outcomes in complex biological systems.

Fourier transform infrared imaging microspectroscopy and tissue-level mechanical testing reveal intraspecies variation in mouse bone mineral and matrix composition.

Fracture susceptibility is heritable and dependent upon bone morphology and quality. However, studies of bone quality are typically overshadowed by emphasis on bone geometry and bone mineral density. Given that differences in mineral and matrix composition exist in a variety of species, we hypothesized that genetic variation in bone quality and tissue-level mechanical properties would also exist within species. Sixteen-week-old female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mouse femora were analyzed using Fourier transform infrared imaging and tissue-level mechanical testing for variation in mineral composition, mineral maturity, collagen cross-link ratio, and tissue-level mechanical properties. A/J femora had an increased mineral-to-matrix ratio compared to B6. The C3H mineral-to-matrix ratio was intermediate of A/J and B6. C3H femora had reduced acid phosphate and carbonate levels and an increased collagen cross-link ratio compared to A/J and B6. Modulus values paralleled mineral-to-matrix values, with A/J femora being the most stiff, B6 being the least stiff, and C3H having intermediate stiffness. In addition, work-to-failure varied among the strains, with the highly mineralized and brittle A/J femora performing the least amount of work-to-failure. Inbred mice are therefore able to differentially modulate the composition of their bone mineral and the maturity of their bone matrix in conjunction with tissue-level mechanical properties. These results suggest that specific combinations of bone quality and morphological traits are genetically regulated such that mechanically functional bones can be constructed in different ways.

Defining nasal cartilage elasticity: biomechanical testing of the tripod theory based on a cantilevered model.

OBJECTIVE: To define the modulus of elasticity for nasal septum, auricular, upper lateral, and lower lateral cartilages. METHODS: Prospective enrollment of sequential patients undergoing septorhinoplasty. Test samples were obtained through routine surgical interventions using atraumatic harvesting techniques. The modulus of elasticity was determined using a customized biomechanical testing device. A clinical analysis of nasal tip strength and "ethnic" nasal categorization was performed. RESULTS: Five sequential patients were enrolled; 4 underwent biomechanical testing of harvested cartilage. All 4 patients were classified as having a leptor-rhine nasal architecture. The modulus of elasticity for the lower lateral cartilages was 1.82 to 15.28 MPa. Values for auricular, nasal septum, and upper lateral cartilages (medial and caudal) were also determined. CONCLUSIONS: This is the first biomechanical study performed on human auricular, lower lateral, and upper lateral cartilages. The elastic modulus can be determined from samples obtained during routine septorhinoplasty. The modulus of elasticity for all areas was significantly higher than values previously demonstrated for bioengineered elastic cartilage and carved human nasal septal specimens. Shaving the lateral portions of the nasal septum may significantly reduce tensile strength, which may affect graft performance in vivo. Further refinement of testing methods and an increase in the number of analyzed samples are required for formal statistical analysis and further determination of clinical relevance in different nasal subtypes.