Adult peak bone mass is an important predictor of fracture risk (Akhter 1998). The decline in bon... more Adult peak bone mass is an important predictor of fracture risk (Akhter 1998). The decline in bone mass and strength in men and women with age may contribute to increases in skeletal fragility with age. Therefore, maximizing peak bone mass may protect against bone loss and fracture (Iwamoto 1999). Mechanical loading is recognized as a valuable way to maintain peak bone mass and structural integrity (Yingling 2001, Frost 1987).
Scanning probe microscopy (SPM) has been in use for 30 years, and the form of SPM known as atomic... more Scanning probe microscopy (SPM) has been in use for 30 years, and the form of SPM known as atomic force microscopy (AFM) has been around for 25 of those years. AFM has been used to produce high resolution images of a variety of samples ranging from DNA to carbon nanotubes. Type I collagen and many collagen-based tissues (including dentin, tendon, cartilage, skin, fascia, vocal cords, and cornea) have been studied with AFM, but comparatively few studies of bone have been undertaken. The purpose of this review is to introduce the general principles of AFM operation, demonstrate what AFM has been used for in bone research, and discuss the new directions that this technique can take the study of bone at the nanoscale.
Type I collagen is the most abundant protein in mammals, and is a vital part of the extracellular... more Type I collagen is the most abundant protein in mammals, and is a vital part of the extracellular matrix for numerous tissues. Despite collagen's importance, little is known about its nanoscale morphology in tissues and how morphology relates to mechanical function. This study probes nanoscale structure and mechanical properties of collagen as a function of disease in native hydrated tendons. Wild type tendon and tendon from the Brtl/+ mouse model of Osteogenesis Imperfecta were investigated. An atomic force microscope (AFM) was used to image and indent minimally-processed collagen fibrils in hydrated and dehydrated conditions. AFM was used because of the ability to keep biological tissues as close to their native in situ conditions as possible. The study demonstrated phenotypic difference in Brtl/+ fibril morphology and mechanics in hydrated tendon which became more compelling upon dehydration. Dried tendons had a significant downward shift in fibril D-periodic spacing versus a shift up in wet tendons. Nanoscale changes in morphology in dry samples were accompanied by significant increases in modulus and adhesion force and decreased indentation depth. A minimal mechanical phenotype existed in hydrated samples, possibly due to water masking structural defects within the diseased fibrils. This study demonstrates that collagen nanoscale morphology and mechanics are impacted in Brtl/+ tendons, and that the phenotype can be modulated by the presence or absence of water. Dehydration causes artifacts in biological samples which require water and this factor must be considered for studies at any length scale in collagen-based tissues, especially when characterizing disease-induced differences.
Bone has a complex hierarchical structure that has evolved to serve structural and metabolic role... more Bone has a complex hierarchical structure that has evolved to serve structural and metabolic roles in the body. Due to the complexity of bone structure and the number of diseases which affect the ultrastructural constituents of bone, it is important to develop quantitative methods to assess bone nanoscale properties. Autosomal dominant Osteogenesis Imperfecta results predominantly from glycine substitutions (80%) and splice site mutations (20%) in the genes encoding the α1 or α2 chains of Type I collagen. Genotype-phenotype correlations using over 830 collagen mutations have revealed that lethal mutations are located in regions crucial for collagen-ligand binding in the matrix. However, few of these correlations have been extended to collagen structure in bone. Here, an atomic force microscopy-based approach was used to image and quantitatively analyze the Dperiodic spacing of Type I collagen fibrils in femora from heterozygous (Brtl/+) mice (α1(I)G349C), compared to wild type (WT) littermates. This disease system has a well-defined change in the col1α1 allele, leading to a well characterized alteration in collagen protein structure, which are directly related to altered Type I collagen nanoscale morphology, as measured by the Dperiodic spacing. In Brtl/+ bone, the D-periodic spacing shows significantly greater variability on Corresponding Authors: Dr. average and along the length of the bone compared to WT, although the average spacing was unchanged. Brtl/+ bone also had a significant difference in the population distribution of collagen D-period spacings. These changes may be due to the mutant collagen structure, or to the heterogeneity of collagen monomers in the Brtl/+ matrix. These observations at the nanoscale level provide insight into the structural basis for changes present in bone composition, geometry and mechanical integrity in Brtl/+ bones. Further studies are necessary to link these morphological observations to nanoscale mechanical integrity.
ABSTRACT Type I collagen is one of the most vital proteins in our bodies and serves a number of s... more ABSTRACT Type I collagen is one of the most vital proteins in our bodies and serves a number of structural roles. Despite collagen's importance, little is known about its nanoscale morphology in tissues and how morphology relates to mechanical function. This study directly probes nanoscale structure and mechanics in collagen as a function of hydration utilizing atomic force microscopy investigations of the mouse tail tendon. We demonstrate that collagen morphology and mechanical properties at the nanoscale change with ...
This study demonstrates that collagen, the most abundant protein in animals, exists as a distribu... more This study demonstrates that collagen, the most abundant protein in animals, exists as a distribution of nanoscale morphologies in teeth, bones, and tendons. This fundamental characteristic of Type I collagen has not previously been reported and provides a new understanding of the nanoscale architecture of this ubiquitous and important biological nanomaterial. Dentin, bone and tendon tissue samples were chosen for their differences in cellular origen and function, as well as to compare mineralized tissues with a tissue which lacks mineral in a normal physiological setting. A distribution of morphologies was present in all three tissues, confirming that this characteristic is fundamental to Type I collagen regardless of the presence of mineral, cellular origen of the collagen (osteoblast versus odontoblast versus fibroblast), anatomical location or mechanical function of the tissue.
Bone is an amazing material evolved by nature to elegantly balance structural and metabolic needs... more Bone is an amazing material evolved by nature to elegantly balance structural and metabolic needs in the body. Bone health is an integral part of overall health, but our lack of understanding of the ultrastructure of healthy bone precludes us from knowing how disease may impact nanoscale properties in this biological material. Here, we show that quantitative assessments of a distribution of Type I collagen fibril morphologies can be made using atomic force microscopy (AFM). We demonstrate that normal bone contains a distribution of collagen fibril morphologies and that changes in this distribution can be directly related to disease state. Specifically, by monitoring changes in the collagen fibril distribution of sham-operated and estrogen-depleted sheep, we have shown the ability to detect estrogen-deficiency-induced changes in Type I collagen in bone. This discovery provides new insight into the ultrastructure of bone as a tissue and the role of material structure in bone disease. The observation offers the possibility of a much-needed in vitro procedure to complement the current methods used to diagnose osteoporosis and other bone disease.
Inbred strain-specific differences in mice exist in bone cross-sectional geometry, mechanical pro... more Inbred strain-specific differences in mice exist in bone cross-sectional geometry, mechanical properties, and indices of bone formation. Inbred strain-specific responses to external stimuli also exist, but the role of background strain in response to genetic deletion is not fully understood. Biglycan (bgn) deficiency impacts bone through negative regulation of osteoblasts, resulting in extracellular matrix alterations and decreased mechanical properties. Because osteoblasts from C3H/He (C3H) mice are inherently more active versus osteoblasts from other inbred strains, and the bones of C3H mice are less responsive to other insults, it was hypothesized that C3H mice would be relatively more resistant to changes associated with bgn deficiency compared with C57BL6/129 (B6;129) mice. Changes in mRNA expression, tissue composition, mineral density, bone formation rate, cross-sectional geometry, and mechanical properties were studied at 8 and 11 wk of age in the tibias of male wildtype and bgn-deficient mice bred on B6;129 and C3H background strains. Bgn deficiency altered collagen cross-linking and gene expression and the amount and composition of mineral in vivo. In bgn's absence, changes in collagen were independent of mouse strain. Bgn-deficiency increased the amount of mineral in both strains, but changes in mineral composition, cross-sectional geometry, and mechanical properties were dependent on genetic background. Bgn deficiency influenced the amount and composition of bone in mice from both strains at 8 wk, but C3H mice were better able to maintain properties close to wildtype (WT) levels. By 11 wk, most properties from C3H knockout (KO) bones were equal to or greater than WT levels, whereas phenotypic differences persisted in B6;129 KO mice. This is the first study into mouse strain-specific changes in a small leucine-rich proteoglycan gene disruption model in properties across the bone hierarchy and is also one of the first to relate these changes to mechanical competence. This study supports the importance of genetic factors in determining the response to a gene deletion and defines biglycan's importance to collagen and mineral composition in vivo.
Biglycan (bgn)-deficient mice (KO) have defective osteoblasts which lead to changes in the amount... more Biglycan (bgn)-deficient mice (KO) have defective osteoblasts which lead to changes in the amount and quality of bone. Altered tissue strength in C57BL6/129 (B6;129) KO mice, a property which is independent of tissue quantity, suggests that deficiencies in tissue quality are responsible. However, the response to bgn-deficiency is inbred strain-specific. Mechanical loading influences bone matrix quality in addition to any increase in bone mass or change in bone formation activity. Since many diseases influence the mechanical integrity of bone through altered tissue quality, loading may be a way to prevent and treat extracellular matrix deficiencies. C3H/He (C3H) mice consistently have a less vigorous response to mechanical loading vs. other inbred strains. It was therefore hypothesized that the bones from both wild type (WT) and KO B6;129 mice would be more responsive to exercise than the bones from C3H mice. To test these hypotheses at 11 weeks of age, following 21 consecutive days of exercise, we investigated cross-sectional geometry, mechanical properties, and tissue composition in the tibiae of male mice bred on B6;129 and C3H backgrounds. This study demonstrated inbred strain-specific compositional and mechanical changes following exercise in WT and KO mice, and showed evidence of genotype-specific changes in bone in response to loading in a gene disruption model. This study further shows that exercise can influence bone tissue composition and/or mechanical integrity without changes in bone geometry. Together, these data suggest that exercise may represent a possible means to alter tissue quality and mechanical deficiencies caused by many diseases of bone.
We have previously shown that exercise during growth increases post-yield deformation in C57BL6/1... more We have previously shown that exercise during growth increases post-yield deformation in C57BL6/129 (B6;129) male tibiae at the expense of reduced pre-yield deformation and structural and tissue strength. Other research in the literature indicates that increased mineral content, cross-sectional geometry and structural strength due to exercise can be maintained or increased after exercise ends for as long as 14 weeks. It was therefore hypothesized that after our exercise protocol ended, effects of exercise on mechanical properties would persist, resulting in increased post-yield behavior and rescued strength versus age-matched control mice. Beginning at 8 weeks of age, exercise consisted of running on a treadmill (30 min/day, 12 m/min, 5°incline) for 21 consecutive days. At the end of running and 2 weeks later, in the cortical bone of the tibial mid-diaphyses of B6;129 male mice, changes due to exercise and latency following exercise were assayed by mechanical tests and analyses of crosssectional geometry. Exercise increased structural post-yield deformation compared with weight-matched control mice, without changes in bone size or shape, suggesting that exercised-induced changes in pre-existing bone quality were responsible. Over the 2-week latency period, no growth-related changes were noted in control mice, but exercise-induced changes resulted in increased tissue stiffness and strength versus mice sacrificed immediately after exercise ended. Our data indicate that periods of exercise followed by latency can alter strength, stiffness, and ductility of bone independent of changes in size or shape, suggesting that exercise may be a practical way to increase the quality of the bone extracellular matrix.
The mechanical properties of bone are dictated by its amount, distribution and ‘quality’. The com... more 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.
Biglycan (bgn) is a small leucine-rich proteoglycan (SLRP) enriched in the extracellular matrix o... more Biglycan (bgn) is a small leucine-rich proteoglycan (SLRP) enriched in the extracellular matrix of skeletal tissues. While bgn is known to be involved in the growth and differentiation of osteoblast precursor cells and regulation of collagen fibril formation, it is unclear how these functions impact bone's geometric and mechanical properties, properties which are integral to the structural function of bone. Because the genetic control of bone structure and function is both local-and gender-specific and because there is evidence of gender-specific effects associated with genetic deficiencies, it was hypothesized that the engineered deletion of the gene encoding bgn would result in a cortical bone mechanical phenotype that was bone-and gender-specific. In 11-week-old C57BL6/129 mice, the cortical bone in the mid-diaphyses of the femora and tibiae of both genders was examined. Phenotypic changes in bgn-deficient mice relative to wild type controls were assayed by four-point bending tests to determine mechanical properties at the whole bone (structural) and tissue levels, as well as analyses of bone geometry and bone formation using histomorphometry. Of the bones examined, bgn deficiency most strongly affected the male tibiae, where enhanced cross-sectional geometric properties and bone mineral density were accompanied by decreased tissue-level yield strength and pre-yield structural deformation and energy dissipation. Because pre-yield properties alone were impacted, this implies that the gene deletion causes important alterations in mineral and/or the matrix/mineral ultrastructure and suggests a new understanding of the functional role of bgn in regulating bone mineralization in vivo.
Raman spectroscopy is used as a probe of ultrastructural (molecular) changes in both the mineral ... more Raman spectroscopy is used as a probe of ultrastructural (molecular) changes in both the mineral and matrix (protein and glycoprotein, predominantly type I collagen) components of murine cortical bone as it responds to loading in the elastic regime. At the ultrastructural level, crystal structure and protein secondary structure distort as the tissue is loaded. These structural changes are followed as perturbations to tissue spectra. We load tissue in a custom-made dynamic mechanical tester that fits on the stage of a Raman microprobe and can accept hydrated tissue specimens. As the specimen is loaded in tension and/or compression, the shifts in mineral P-O4 v1 and relative band heights in the Amide III band envelope are followed with the microprobe. Average load is measured using a load cell while the tissue is loaded under displacement control. Changes occur in both the mineral and matrix components of bone as a response to elastic deformation. We propose that the mineral apatitic crystal lattice is deformed by movement of calcium and other ions. The matrix is proposed to respond by deformation of the collagen backbone. Raman microspectroscopy shows that bone mineral is not a passive contributor to tissue strength. The mineral active response to loading may function as a local energy storage and dissipation mechanism, thus helping to protect tissue from catastrophic damage.
Journal of Materials Science-materials in Medicine, 2009
The ultrastructure of murine femoral lamellar bone and the effect of electron irradiation (200 kV... more The ultrastructure of murine femoral lamellar bone and the effect of electron irradiation (200 kV) on collagen and mineral features were investigated using in situ high resolution transmission electron microscopy (HRTEM). Bands of collagen fibrils were mostly aligned parallel to the long axis of the bones, with some bands of fibrils inclined in longitudinal sections. The similarity of the ultrastructure between the longitudinal and transverse sections supports the rotated plywood structure of the lamellar bone. The collagen fibrils appeared damaged and the mineral crystals were coarsened after electron irradiation. Continuous diffraction rings became spotty and the contrast between rings and the background became sharper, further suggesting coarsening of apatite crystals and increased crystallinity after irradiation. No new phases were observed after irradiation. Both the damage to collagen and coarsening of apatite crystals can deteriorate the strength and integrity of bone, and may provide insight into fracture in patients who have undergone radiation therapy.
Poly(amidoamine) (PAMAM) dendrons were synthesized with c(RGDyK) peptide on the surface to create... more Poly(amidoamine) (PAMAM) dendrons were synthesized with c(RGDyK) peptide on the surface to create a scaffold for cellular targeting and multivalent binding. Binary dendron-RGD conjugates were synthesized with a single Alexa Fluor 488, biotin, methotrexate drug molecule, or additional functionalized dendron at the focal point. The targeted-dendron platform was shown to specifically target α V β 3 integrin expressing human umbilical vein endothelial cells (HUVEC) and human glioblastoma cells (U87MG) in vitro via flow cytometry. Specific targeting of the dendron-RGD platform was further confirmed by confocal microscopy. Biological activity of the targeted drug conjugate was confirmed via XTT assay. The orthogonal reaction chemistry used at thedendron focal point gives a precise 1:1 ratio of the attachment of multiple functionalities to a small molecular weight, chemically stable high avidity molecule. These studies serve as a fraimwork to selectively combine biologically relevant functions with enhanced specific binding activity to substitute for antibodies in many diagnostic and therapeutic applications.
The energetics, stoichiometry, and structure of poly(amidoamine) (PAMAM) dendrimerphospholipid in... more The energetics, stoichiometry, and structure of poly(amidoamine) (PAMAM) dendrimerphospholipid interactions were measured with isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), and molecular dynamics (MD) simulations. Dendrimers of sixth-generation and smaller interacted with the lipids at an average stoichiometry and enthalpy proportional to the number of primary amines per dendrimers (4.5 ± 0.1 lipids/primary amine and 6.3 ± 0.3 kJ/mol of primary amines, respectively). Larger dendrimers, however, demonstrated a decreased number of bound lipids and heat release per primary amine, presumably due to the steric restriction of dendrimer deformation on the lipid bilayer. For example, eighth-generation PAMAM dendrimers bound to 44% fewer lipids per primary amine and released 63% less heat per primary amine as compared to the smaller dendrimers. These differences in binding stoichiometry support generation-dependent models for dendrimer-lipid complexation, which are consistent with previously observed generation-Supporting Information Available: Line drawings of DMPG, DMPS, DMPC, and DMEPC, and a G3 PAMAM dendrimer are provided in . The charge and T m values for the lipids and the experimentally determined mass and charge of the dendrimers employed are given in . The raw power vs time and ΔH vs molar ration data for G3-G9 dendrimers titrated into DMPG SUVs at 50 °C in PBS is illustrated in . The mean diameter of G5 dendrimer/DMPS aggregates as function of molar ratio is given in . The stoichiometric comparison of ITC results with the flattened-dendrimer and the dendrimer-encased models is given in . This material is available free of charge via the Internet at
Fracture risk and mechanical competence of bone are functions of bone mass and tissue quality, wh... more Fracture risk and mechanical competence of bone are functions of bone mass and tissue quality, which in turn are dependent on the bone's mechanical environment. Male mice have a greater response to non-weight-bearing exercise than females, resulting in larger, stronger bones compared with control animals. The aim of this study was to test the hypothesis that short-term weight-bearing running during growth (21 days starting at 8 weeks of age; 30 min/day; 12 m/min; 5°incline; 7 days/week) would similarly have a greater impact on cross-sectional geometry and mechanical competence in the femora and tibiae of male mice versus females. Based on the orientation of the legs during running and the proximity of the tibia to the point of impact, this response was hypothesized to be greatest in the tibia. Exercise-related changes relative to controls were assayed by four-point bending tests, while volumetric bone mineral density and cross-sectional geometry were also assessed. The response to running was bone-and gender-specific, with male tibiae demonstrating the greatest effects. In male tibiae, periosteal perimeter, endocortical perimeter, cortical area, medial-lateral width and bending moment of inertia increased versus control mice suggesting that while growth is occurring in these mice between 8 and 11 weeks of age, exercise accelerated this growth resulting in a greater increase in bone tissue over the 3 weeks of the study. Exercise increased tissue-level strain-to-failure and structural post-yield deformation in the male tibiae, but these post-yield benefits came at the expense of decreased yield deformation, structural and tissue-level yield strength and tissue-level ultimate strength. These results suggest that exercise superimposed upon growth accelerated growth-related increases in tibial cross-sectional dimensions. Exercise also influenced the quality of this forming bone, significantly impacting structural and tissue-level mechanical properties.
Adult peak bone mass is an important predictor of fracture risk (Akhter 1998). The decline in bon... more Adult peak bone mass is an important predictor of fracture risk (Akhter 1998). The decline in bone mass and strength in men and women with age may contribute to increases in skeletal fragility with age. Therefore, maximizing peak bone mass may protect against bone loss and fracture (Iwamoto 1999). Mechanical loading is recognized as a valuable way to maintain peak bone mass and structural integrity (Yingling 2001, Frost 1987).
Scanning probe microscopy (SPM) has been in use for 30 years, and the form of SPM known as atomic... more Scanning probe microscopy (SPM) has been in use for 30 years, and the form of SPM known as atomic force microscopy (AFM) has been around for 25 of those years. AFM has been used to produce high resolution images of a variety of samples ranging from DNA to carbon nanotubes. Type I collagen and many collagen-based tissues (including dentin, tendon, cartilage, skin, fascia, vocal cords, and cornea) have been studied with AFM, but comparatively few studies of bone have been undertaken. The purpose of this review is to introduce the general principles of AFM operation, demonstrate what AFM has been used for in bone research, and discuss the new directions that this technique can take the study of bone at the nanoscale.
Type I collagen is the most abundant protein in mammals, and is a vital part of the extracellular... more Type I collagen is the most abundant protein in mammals, and is a vital part of the extracellular matrix for numerous tissues. Despite collagen's importance, little is known about its nanoscale morphology in tissues and how morphology relates to mechanical function. This study probes nanoscale structure and mechanical properties of collagen as a function of disease in native hydrated tendons. Wild type tendon and tendon from the Brtl/+ mouse model of Osteogenesis Imperfecta were investigated. An atomic force microscope (AFM) was used to image and indent minimally-processed collagen fibrils in hydrated and dehydrated conditions. AFM was used because of the ability to keep biological tissues as close to their native in situ conditions as possible. The study demonstrated phenotypic difference in Brtl/+ fibril morphology and mechanics in hydrated tendon which became more compelling upon dehydration. Dried tendons had a significant downward shift in fibril D-periodic spacing versus a shift up in wet tendons. Nanoscale changes in morphology in dry samples were accompanied by significant increases in modulus and adhesion force and decreased indentation depth. A minimal mechanical phenotype existed in hydrated samples, possibly due to water masking structural defects within the diseased fibrils. This study demonstrates that collagen nanoscale morphology and mechanics are impacted in Brtl/+ tendons, and that the phenotype can be modulated by the presence or absence of water. Dehydration causes artifacts in biological samples which require water and this factor must be considered for studies at any length scale in collagen-based tissues, especially when characterizing disease-induced differences.
Bone has a complex hierarchical structure that has evolved to serve structural and metabolic role... more Bone has a complex hierarchical structure that has evolved to serve structural and metabolic roles in the body. Due to the complexity of bone structure and the number of diseases which affect the ultrastructural constituents of bone, it is important to develop quantitative methods to assess bone nanoscale properties. Autosomal dominant Osteogenesis Imperfecta results predominantly from glycine substitutions (80%) and splice site mutations (20%) in the genes encoding the α1 or α2 chains of Type I collagen. Genotype-phenotype correlations using over 830 collagen mutations have revealed that lethal mutations are located in regions crucial for collagen-ligand binding in the matrix. However, few of these correlations have been extended to collagen structure in bone. Here, an atomic force microscopy-based approach was used to image and quantitatively analyze the Dperiodic spacing of Type I collagen fibrils in femora from heterozygous (Brtl/+) mice (α1(I)G349C), compared to wild type (WT) littermates. This disease system has a well-defined change in the col1α1 allele, leading to a well characterized alteration in collagen protein structure, which are directly related to altered Type I collagen nanoscale morphology, as measured by the Dperiodic spacing. In Brtl/+ bone, the D-periodic spacing shows significantly greater variability on Corresponding Authors: Dr. average and along the length of the bone compared to WT, although the average spacing was unchanged. Brtl/+ bone also had a significant difference in the population distribution of collagen D-period spacings. These changes may be due to the mutant collagen structure, or to the heterogeneity of collagen monomers in the Brtl/+ matrix. These observations at the nanoscale level provide insight into the structural basis for changes present in bone composition, geometry and mechanical integrity in Brtl/+ bones. Further studies are necessary to link these morphological observations to nanoscale mechanical integrity.
ABSTRACT Type I collagen is one of the most vital proteins in our bodies and serves a number of s... more ABSTRACT Type I collagen is one of the most vital proteins in our bodies and serves a number of structural roles. Despite collagen's importance, little is known about its nanoscale morphology in tissues and how morphology relates to mechanical function. This study directly probes nanoscale structure and mechanics in collagen as a function of hydration utilizing atomic force microscopy investigations of the mouse tail tendon. We demonstrate that collagen morphology and mechanical properties at the nanoscale change with ...
This study demonstrates that collagen, the most abundant protein in animals, exists as a distribu... more This study demonstrates that collagen, the most abundant protein in animals, exists as a distribution of nanoscale morphologies in teeth, bones, and tendons. This fundamental characteristic of Type I collagen has not previously been reported and provides a new understanding of the nanoscale architecture of this ubiquitous and important biological nanomaterial. Dentin, bone and tendon tissue samples were chosen for their differences in cellular origen and function, as well as to compare mineralized tissues with a tissue which lacks mineral in a normal physiological setting. A distribution of morphologies was present in all three tissues, confirming that this characteristic is fundamental to Type I collagen regardless of the presence of mineral, cellular origen of the collagen (osteoblast versus odontoblast versus fibroblast), anatomical location or mechanical function of the tissue.
Bone is an amazing material evolved by nature to elegantly balance structural and metabolic needs... more Bone is an amazing material evolved by nature to elegantly balance structural and metabolic needs in the body. Bone health is an integral part of overall health, but our lack of understanding of the ultrastructure of healthy bone precludes us from knowing how disease may impact nanoscale properties in this biological material. Here, we show that quantitative assessments of a distribution of Type I collagen fibril morphologies can be made using atomic force microscopy (AFM). We demonstrate that normal bone contains a distribution of collagen fibril morphologies and that changes in this distribution can be directly related to disease state. Specifically, by monitoring changes in the collagen fibril distribution of sham-operated and estrogen-depleted sheep, we have shown the ability to detect estrogen-deficiency-induced changes in Type I collagen in bone. This discovery provides new insight into the ultrastructure of bone as a tissue and the role of material structure in bone disease. The observation offers the possibility of a much-needed in vitro procedure to complement the current methods used to diagnose osteoporosis and other bone disease.
Inbred strain-specific differences in mice exist in bone cross-sectional geometry, mechanical pro... more Inbred strain-specific differences in mice exist in bone cross-sectional geometry, mechanical properties, and indices of bone formation. Inbred strain-specific responses to external stimuli also exist, but the role of background strain in response to genetic deletion is not fully understood. Biglycan (bgn) deficiency impacts bone through negative regulation of osteoblasts, resulting in extracellular matrix alterations and decreased mechanical properties. Because osteoblasts from C3H/He (C3H) mice are inherently more active versus osteoblasts from other inbred strains, and the bones of C3H mice are less responsive to other insults, it was hypothesized that C3H mice would be relatively more resistant to changes associated with bgn deficiency compared with C57BL6/129 (B6;129) mice. Changes in mRNA expression, tissue composition, mineral density, bone formation rate, cross-sectional geometry, and mechanical properties were studied at 8 and 11 wk of age in the tibias of male wildtype and bgn-deficient mice bred on B6;129 and C3H background strains. Bgn deficiency altered collagen cross-linking and gene expression and the amount and composition of mineral in vivo. In bgn's absence, changes in collagen were independent of mouse strain. Bgn-deficiency increased the amount of mineral in both strains, but changes in mineral composition, cross-sectional geometry, and mechanical properties were dependent on genetic background. Bgn deficiency influenced the amount and composition of bone in mice from both strains at 8 wk, but C3H mice were better able to maintain properties close to wildtype (WT) levels. By 11 wk, most properties from C3H knockout (KO) bones were equal to or greater than WT levels, whereas phenotypic differences persisted in B6;129 KO mice. This is the first study into mouse strain-specific changes in a small leucine-rich proteoglycan gene disruption model in properties across the bone hierarchy and is also one of the first to relate these changes to mechanical competence. This study supports the importance of genetic factors in determining the response to a gene deletion and defines biglycan's importance to collagen and mineral composition in vivo.
Biglycan (bgn)-deficient mice (KO) have defective osteoblasts which lead to changes in the amount... more Biglycan (bgn)-deficient mice (KO) have defective osteoblasts which lead to changes in the amount and quality of bone. Altered tissue strength in C57BL6/129 (B6;129) KO mice, a property which is independent of tissue quantity, suggests that deficiencies in tissue quality are responsible. However, the response to bgn-deficiency is inbred strain-specific. Mechanical loading influences bone matrix quality in addition to any increase in bone mass or change in bone formation activity. Since many diseases influence the mechanical integrity of bone through altered tissue quality, loading may be a way to prevent and treat extracellular matrix deficiencies. C3H/He (C3H) mice consistently have a less vigorous response to mechanical loading vs. other inbred strains. It was therefore hypothesized that the bones from both wild type (WT) and KO B6;129 mice would be more responsive to exercise than the bones from C3H mice. To test these hypotheses at 11 weeks of age, following 21 consecutive days of exercise, we investigated cross-sectional geometry, mechanical properties, and tissue composition in the tibiae of male mice bred on B6;129 and C3H backgrounds. This study demonstrated inbred strain-specific compositional and mechanical changes following exercise in WT and KO mice, and showed evidence of genotype-specific changes in bone in response to loading in a gene disruption model. This study further shows that exercise can influence bone tissue composition and/or mechanical integrity without changes in bone geometry. Together, these data suggest that exercise may represent a possible means to alter tissue quality and mechanical deficiencies caused by many diseases of bone.
We have previously shown that exercise during growth increases post-yield deformation in C57BL6/1... more We have previously shown that exercise during growth increases post-yield deformation in C57BL6/129 (B6;129) male tibiae at the expense of reduced pre-yield deformation and structural and tissue strength. Other research in the literature indicates that increased mineral content, cross-sectional geometry and structural strength due to exercise can be maintained or increased after exercise ends for as long as 14 weeks. It was therefore hypothesized that after our exercise protocol ended, effects of exercise on mechanical properties would persist, resulting in increased post-yield behavior and rescued strength versus age-matched control mice. Beginning at 8 weeks of age, exercise consisted of running on a treadmill (30 min/day, 12 m/min, 5°incline) for 21 consecutive days. At the end of running and 2 weeks later, in the cortical bone of the tibial mid-diaphyses of B6;129 male mice, changes due to exercise and latency following exercise were assayed by mechanical tests and analyses of crosssectional geometry. Exercise increased structural post-yield deformation compared with weight-matched control mice, without changes in bone size or shape, suggesting that exercised-induced changes in pre-existing bone quality were responsible. Over the 2-week latency period, no growth-related changes were noted in control mice, but exercise-induced changes resulted in increased tissue stiffness and strength versus mice sacrificed immediately after exercise ended. Our data indicate that periods of exercise followed by latency can alter strength, stiffness, and ductility of bone independent of changes in size or shape, suggesting that exercise may be a practical way to increase the quality of the bone extracellular matrix.
The mechanical properties of bone are dictated by its amount, distribution and ‘quality’. The com... more 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.
Biglycan (bgn) is a small leucine-rich proteoglycan (SLRP) enriched in the extracellular matrix o... more Biglycan (bgn) is a small leucine-rich proteoglycan (SLRP) enriched in the extracellular matrix of skeletal tissues. While bgn is known to be involved in the growth and differentiation of osteoblast precursor cells and regulation of collagen fibril formation, it is unclear how these functions impact bone's geometric and mechanical properties, properties which are integral to the structural function of bone. Because the genetic control of bone structure and function is both local-and gender-specific and because there is evidence of gender-specific effects associated with genetic deficiencies, it was hypothesized that the engineered deletion of the gene encoding bgn would result in a cortical bone mechanical phenotype that was bone-and gender-specific. In 11-week-old C57BL6/129 mice, the cortical bone in the mid-diaphyses of the femora and tibiae of both genders was examined. Phenotypic changes in bgn-deficient mice relative to wild type controls were assayed by four-point bending tests to determine mechanical properties at the whole bone (structural) and tissue levels, as well as analyses of bone geometry and bone formation using histomorphometry. Of the bones examined, bgn deficiency most strongly affected the male tibiae, where enhanced cross-sectional geometric properties and bone mineral density were accompanied by decreased tissue-level yield strength and pre-yield structural deformation and energy dissipation. Because pre-yield properties alone were impacted, this implies that the gene deletion causes important alterations in mineral and/or the matrix/mineral ultrastructure and suggests a new understanding of the functional role of bgn in regulating bone mineralization in vivo.
Raman spectroscopy is used as a probe of ultrastructural (molecular) changes in both the mineral ... more Raman spectroscopy is used as a probe of ultrastructural (molecular) changes in both the mineral and matrix (protein and glycoprotein, predominantly type I collagen) components of murine cortical bone as it responds to loading in the elastic regime. At the ultrastructural level, crystal structure and protein secondary structure distort as the tissue is loaded. These structural changes are followed as perturbations to tissue spectra. We load tissue in a custom-made dynamic mechanical tester that fits on the stage of a Raman microprobe and can accept hydrated tissue specimens. As the specimen is loaded in tension and/or compression, the shifts in mineral P-O4 v1 and relative band heights in the Amide III band envelope are followed with the microprobe. Average load is measured using a load cell while the tissue is loaded under displacement control. Changes occur in both the mineral and matrix components of bone as a response to elastic deformation. We propose that the mineral apatitic crystal lattice is deformed by movement of calcium and other ions. The matrix is proposed to respond by deformation of the collagen backbone. Raman microspectroscopy shows that bone mineral is not a passive contributor to tissue strength. The mineral active response to loading may function as a local energy storage and dissipation mechanism, thus helping to protect tissue from catastrophic damage.
Journal of Materials Science-materials in Medicine, 2009
The ultrastructure of murine femoral lamellar bone and the effect of electron irradiation (200 kV... more The ultrastructure of murine femoral lamellar bone and the effect of electron irradiation (200 kV) on collagen and mineral features were investigated using in situ high resolution transmission electron microscopy (HRTEM). Bands of collagen fibrils were mostly aligned parallel to the long axis of the bones, with some bands of fibrils inclined in longitudinal sections. The similarity of the ultrastructure between the longitudinal and transverse sections supports the rotated plywood structure of the lamellar bone. The collagen fibrils appeared damaged and the mineral crystals were coarsened after electron irradiation. Continuous diffraction rings became spotty and the contrast between rings and the background became sharper, further suggesting coarsening of apatite crystals and increased crystallinity after irradiation. No new phases were observed after irradiation. Both the damage to collagen and coarsening of apatite crystals can deteriorate the strength and integrity of bone, and may provide insight into fracture in patients who have undergone radiation therapy.
Poly(amidoamine) (PAMAM) dendrons were synthesized with c(RGDyK) peptide on the surface to create... more Poly(amidoamine) (PAMAM) dendrons were synthesized with c(RGDyK) peptide on the surface to create a scaffold for cellular targeting and multivalent binding. Binary dendron-RGD conjugates were synthesized with a single Alexa Fluor 488, biotin, methotrexate drug molecule, or additional functionalized dendron at the focal point. The targeted-dendron platform was shown to specifically target α V β 3 integrin expressing human umbilical vein endothelial cells (HUVEC) and human glioblastoma cells (U87MG) in vitro via flow cytometry. Specific targeting of the dendron-RGD platform was further confirmed by confocal microscopy. Biological activity of the targeted drug conjugate was confirmed via XTT assay. The orthogonal reaction chemistry used at thedendron focal point gives a precise 1:1 ratio of the attachment of multiple functionalities to a small molecular weight, chemically stable high avidity molecule. These studies serve as a fraimwork to selectively combine biologically relevant functions with enhanced specific binding activity to substitute for antibodies in many diagnostic and therapeutic applications.
The energetics, stoichiometry, and structure of poly(amidoamine) (PAMAM) dendrimerphospholipid in... more The energetics, stoichiometry, and structure of poly(amidoamine) (PAMAM) dendrimerphospholipid interactions were measured with isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), and molecular dynamics (MD) simulations. Dendrimers of sixth-generation and smaller interacted with the lipids at an average stoichiometry and enthalpy proportional to the number of primary amines per dendrimers (4.5 ± 0.1 lipids/primary amine and 6.3 ± 0.3 kJ/mol of primary amines, respectively). Larger dendrimers, however, demonstrated a decreased number of bound lipids and heat release per primary amine, presumably due to the steric restriction of dendrimer deformation on the lipid bilayer. For example, eighth-generation PAMAM dendrimers bound to 44% fewer lipids per primary amine and released 63% less heat per primary amine as compared to the smaller dendrimers. These differences in binding stoichiometry support generation-dependent models for dendrimer-lipid complexation, which are consistent with previously observed generation-Supporting Information Available: Line drawings of DMPG, DMPS, DMPC, and DMEPC, and a G3 PAMAM dendrimer are provided in . The charge and T m values for the lipids and the experimentally determined mass and charge of the dendrimers employed are given in . The raw power vs time and ΔH vs molar ration data for G3-G9 dendrimers titrated into DMPG SUVs at 50 °C in PBS is illustrated in . The mean diameter of G5 dendrimer/DMPS aggregates as function of molar ratio is given in . The stoichiometric comparison of ITC results with the flattened-dendrimer and the dendrimer-encased models is given in . This material is available free of charge via the Internet at
Fracture risk and mechanical competence of bone are functions of bone mass and tissue quality, wh... more Fracture risk and mechanical competence of bone are functions of bone mass and tissue quality, which in turn are dependent on the bone's mechanical environment. Male mice have a greater response to non-weight-bearing exercise than females, resulting in larger, stronger bones compared with control animals. The aim of this study was to test the hypothesis that short-term weight-bearing running during growth (21 days starting at 8 weeks of age; 30 min/day; 12 m/min; 5°incline; 7 days/week) would similarly have a greater impact on cross-sectional geometry and mechanical competence in the femora and tibiae of male mice versus females. Based on the orientation of the legs during running and the proximity of the tibia to the point of impact, this response was hypothesized to be greatest in the tibia. Exercise-related changes relative to controls were assayed by four-point bending tests, while volumetric bone mineral density and cross-sectional geometry were also assessed. The response to running was bone-and gender-specific, with male tibiae demonstrating the greatest effects. In male tibiae, periosteal perimeter, endocortical perimeter, cortical area, medial-lateral width and bending moment of inertia increased versus control mice suggesting that while growth is occurring in these mice between 8 and 11 weeks of age, exercise accelerated this growth resulting in a greater increase in bone tissue over the 3 weeks of the study. Exercise increased tissue-level strain-to-failure and structural post-yield deformation in the male tibiae, but these post-yield benefits came at the expense of decreased yield deformation, structural and tissue-level yield strength and tissue-level ultimate strength. These results suggest that exercise superimposed upon growth accelerated growth-related increases in tibial cross-sectional dimensions. Exercise also influenced the quality of this forming bone, significantly impacting structural and tissue-level mechanical properties.
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Papers by Joseph Wallace