Metabolic and Skeletal Characterization of the KK/Ay Mouse Model-A Polygenic Mutation Model of Obese Type 2 Diabetes.

Type 2 diabetes (T2D) increases fracture incidence and fracture-related mortality rates (KK.Cg-Ay/J. The Jackson Laboratory; Available from: https://www.jax.org/strain/002468 ). While numerous mouse models for T2D exist, few effectively stimulate persistent hyperglycemia in both sexes, and even fewer are suitable for bone studies. Commonly used models like db/db and ob/ob have altered leptin pathways, confounding bone-related findings since leptin regulates bone properties (Fajardo et al. in Journal of Bone and Mineral Research 29(5): 1025-1040, 2014). The Yellow Kuo Kondo (KK/Ay) mouse, a polygenic mutation model of T2D, is able to produce a consistent diabetic state in both sexes and addresses the lack of a suitable model of T2D for bone studies. The diabetic state of KK/Ay stems from a mutation in the agouti gene, responsible for coat color in mice. This mutation induces ectopic gene expression across various tissue types, resulting in diabetic mice with yellow fur coats (Moussa and Claycombe in Obesity Research 7(5): 506-514, 1999). Male and female KK/Ay mice exhibited persistent hyperglycemia, defining them as diabetic with blood glucose (BG) levels consistently exceeding 300 mg/dL. Notably, male control mice in this study were also diabetic, presenting a significant limitation. Nevertheless, male and female KK/Ay mice showed significantly elevated BG levels, HbA1c, and serum insulin concentration when compared to the non-diabetic female control mice. Early stages of T2D are characterized by hyperglycemia and hyperinsulinemia resulting from cellular insulin resistance, whereas later stages may feature hypoinsulinemia due to ß-cell apoptosis (Banday et al. Avicenna Journal of Medicine 10(04): 174-188, 2020 and Klein et al. Cell Metabolism 34(1): 11-20, 2022). The observed hyperglycemia, hyperinsulinemia, and the absence of differences in ß-cell mass suggest that KK/Ay mice in this study are modeling the earlier stages of T2D. While compromised bone microarchitecture was observed in this study, older KK/Ay mice, representing more advanced stages of T2D, might exhibit more pronounced skeletal manifestations. Compared to the control group, the femora of KK/Ay mice had higher cortical area and cortical thickness, and improved trabecular properties which would typically be indicative of greater bone strength. However, KK/Ay mice displayed lower cortical tissue mineral density in both sexes and increased cortical porosity in females. Fracture instability toughness of the femora was lower in KK/Ay mice overall compared to controls. These findings indicate that decreased mechanical integrity noted in the femora of KK/Ay mice was likely due to overall bone quality being compromised.

Combined Thermoneutral Housing and Raloxifene Treatment Improves Trabecular Bone Microarchitecture and Strength in Growing Female Mice.

Thermoneutral housing and Raloxifene (RAL) treatment both have potential for improving mechanical and architectural properties of bone. Housing mice within a 30 to 32 °C range improves bone quality by reducing the consequences of cold stress, such as shivering and metabolic energy consumption (Chevalier et al. in Cell Metab 32(4):575-590.e7, 2020; Martin et al. in Endocr Connect 8(11):1455-1467, 2019; Hankenson et al. in Comp Med 68(6):425-438, 2018). Previous work suggests that Raloxifene can enhance bone strength and geometry (Ettinger et al. in Jama 282(7):637-645, 1999; Powell et al. in Bone Rep 12:100246, 2020). An earlier study in our lab utilized long bones to examine the effect of thermoneutral housing and Raloxifene treatment in mice, but no significant interactive effects were found. The lack of an impact is hypothesized to be connected to the short 6-week duration of the study and the type of bone analyzed. This study will examine the same question within the axial skeleton, which has a higher proportion of trabecular bone. After 6 weeks of treatment with RAL, vertebrae from female C57BL/6 J mice underwent microcomputed tomography (µCT), architectural analysis, and compression testing. Most of the tested geometric properties (bone volume/tissue volume percent, trabecular thickness, trabecular number, trabecular spacing) improved with both the housing and RAL treatment. The effect sizes suggested an additive effect when treating mice housed under thermoneutral conditions. While ultimate force was enhanced with the treatment and housing, force normalized by bone volume fraction was not significantly different between groups. For longer pre-clinical trials, it may be important to consider the impacts of temperature on mice to improve the accuracy of these models.

NMP4, an Arbiter of Bone Cell Secretory Capacity and Regulator of Skeletal Response to PTH Therapy.

The skeleton is a secretory organ, and the goal of some osteoporosis therapies is to maximize bone matrix output. Nmp4 encodes a novel transcription factor that regulates bone cell secretion as part of its functional repertoire. Loss of Nmp4 enhances bone response to osteoanabolic therapy, in part, by increasing the production and delivery of bone matrix. Nmp4 shares traits with scaling factors, which are transcription factors that influence the expression of hundreds of genes to govern proteome allocation for establishing secretory cell infrastructure and capacity. Nmp4 is expressed in all tissues and while global loss of this gene leads to no overt baseline phenotype, deletion of Nmp4 has broad tissue effects in mice challenged with certain stressors. In addition to an enhanced response to osteoporosis therapies, Nmp4-deficient mice are less sensitive to high fat diet-induced weight gain and insulin resistance, exhibit a reduced disease severity in response to influenza A virus (IAV) infection, and resist the development of some forms of rheumatoid arthritis. In this review, we present the current understanding of the mechanisms underlying Nmp4 regulation of the skeletal response to osteoanabolics, and we discuss how this unique gene contributes to the diverse phenotypes among different tissues and stresses. An emerging theme is that Nmp4 is important for the infrastructure and capacity of secretory cells that are critical for health and disease.

Nmp4, a Regulator of Induced Osteoanabolism, Also Influences Insulin Secretion and Sensitivity.

A bidirectional and complex relationship exists between bone and glycemia. Persons with type 2 diabetes (T2D) are at risk for bone loss and fracture, however, heightened osteoanabolism may ameliorate T2D-induced deficits in glycemia as bone-forming osteoblasts contribute to energy metabolism via increased glucose uptake and cellular glycolysis. Mice globally lacking nuclear matrix protein 4 (Nmp4), a transcription factor expressed in all tissues and conserved between humans and rodents, are healthy and exhibit enhanced bone formation in response to anabolic osteoporosis therapies. To test whether loss of Nmp4 similarly impacted bone deficits caused by diet-induced obesity, male wild-type and Nmp4-/- mice (8 weeks) were fed either low-fat diet or high-fat diet (HFD) for 12 weeks. Endpoint parameters included bone architecture, structural and estimated tissue-level mechanical properties, body weight/composition, glucose-stimulated insulin secretion, glucose tolerance, insulin tolerance, and metabolic cage analysis. HFD diminished bone architecture and ultimate force and stiffness equally in both genotypes. Unexpectedly, the Nmp4-/- mice exhibited deficits in pancreatic ß-cell function and were modestly glucose intolerant under normal diet conditions. Despite the ß-cell deficits, the Nmp4-/- mice were less sensitive to HFD-induced weight gain, increases in % fat mass, and decreases in glucose tolerance and insulin sensitivity. We conclude that Nmp4 supports pancreatic ß-cell function but suppresses peripheral glucose utilization, perhaps contributing to its suppression of induced skeletal anabolism. Selective disruption of Nmp4 in peripheral tissues may provide a strategy for improving both induced osteoanabolism and energy metabolism in comorbid patients.

Effects of Raloxifene and tibial loading on bone mass and mechanics in male and female mice.

Purpose: Raloxifene (RAL) is a selective estrogen receptor modulator (SERM) that has previously been shown to cause acellular benefits to bone tissue. Due to these improvements, RAL was combined with targeted tibial loading to assess if RAL treatment during periods of active bone formation would allow for further mechanical enhancements.Methods: Structural, mechanical, and microstructural effects were assessed in bone from C57BL/6 mice that were treated with RAL (0.5 mg/kg), tibial loading, or both for 6 weeks, beginning at 10 weeks of age.Results:Ex vivo microcomputed tomography (CT) images indicated RAL and loading work together to improve bone mass and architecture, especially within the cancellous region of males. Increases in cancellous bone volume fraction were heavily driven by increases in trabecular thickness, though there were some effects on trabecular spacing and number. In the cortical regions, RAL and loading both increased cross-sectional area, cortical area, and cortical thickness. Whole-bone mechanical testing primarily indicated the effects of loading. Further characterization through Raman spectroscopy and nanoindentation showed load-based changes in mineralization and micromechanics, while both loading and RAL caused changes in the secondary collagen structure. In contrast to males, in females, there were large load-based effects in the cancellous and cortical regions, resulting in increased whole-bone mechanical properties. RAL had less of an effect on cancellous and cortical architecture, though some effects were still present.Conclusion: RAL and loading work together to impact bone architecture and mechanical integrity, leading to greater improvements than either treatment individually.

Iron deficiency and high-intensity running interval training do not impact femoral or tibial bone in young female rats.

In the USA, as many as 20 % of recruits sustain stress fractures during basic training. In addition, approximately one-third of female recruits develop Fe deficiency upon completion of training. Fe is a cofactor in bone collagen formation and vitamin D activation, thus we hypothesised Fe deficiency may be contributing to altered bone microarchitecture and mechanics during 12-weeks of increased mechanical loading. Three-week old female Sprague Dawley rats were assigned to one of four groups: Fe-adequate sedentary, Fe-deficient sedentary, Fe-adequate exercise and Fe-deficient exercise. Exercise consisted of high-intensity treadmill running (54 min 3×/week). After 12-weeks, serum bone turnover markers, femoral geometry and microarchitecture, mechanical properties and fracture toughness and tibiae mineral composition and morphometry were measured. Fe deficiency increased the bone resorption markers C-terminal telopeptide type I collagen and tartate-resistant acid phosphatase 5b (TRAcP 5b). In exercised rats, Fe deficiency further increased bone TRAcP 5b, while in Fe-adequate rats, exercise increased the bone formation marker procollagen type I N-terminal propeptide. In the femur, exercise increased cortical thickness and maximum load. In the tibia, Fe deficiency increased the rate of bone formation, mineral apposition and Zn content. These data show that the femur and tibia structure and mechanical properties are not negatively impacted by Fe deficiency despite a decrease in tibiae Fe content and increase in serum bone resorption markers during 12-weeks of high-intensity running in young growing female rats.

The impact of advanced glycation end products on bone properties in chronic kidney disease.

PURPOSE OF REVIEW: Chronic kidney disease (CKD) affects over 15% of Americans and results in an increased risk of skeletal fractures and fracture-related mortality. However, there remain great challenges in estimating fracture risk in CKD patients, as conventional metrics such as bone density assess bone quantity without accounting for the material quality of the bone tissue. The purpose of this review is to highlight the detrimental effects of advanced glycation end products (AGEs) on the structural and mechanical properties of bone, and to demonstrate the importance of including bone quality when assessing fracture risk in CKD patients. RECENT FINDINGS: Increased oxidative stress and inflammation drive the production of AGEs in CKD patients that form nonenzymatic crosslinks between type I collagen fibrils in the bone matrix. Nonenzymatic crosslinks stiffen and embrittle the bone, reducing its ability to absorb energy and resist fracture. Clinical measurement of AGEs is typically indirect and fails to distinguish the identity and properties of the various AGEs. SUMMARY: Accounting for the impact of AGEs on the skeleton in CKD patients may improve our estimation of overall bone quality, fracture risk, and treatments to improve both bone quantity and quality by reducing AGEs in patients with CKD merit investigation in order to improve our understanding of the etiology of increased fracture risk.

Voluntary Wheel Running Has Beneficial Effects in a Rat Model of CKD-Mineral Bone Disorder (CKD-MBD).

BACKGROUND: Reduced bone and muscle health in individuals with CKD contributes to their higher rates of morbidity and mortality. METHODS: We tested the hypothesis that voluntary wheel running would improve musculoskeletal health in a CKD rat model. Rats with spontaneous progressive cystic kidney disease (Cy/+ IU) and normal littermates (NL) were given access to a voluntary running wheel or standard cage conditions for 10 weeks starting at 25 weeks of age when the rats with kidney disease had reached stage 2-3 of CKD. We then measured the effects of wheel running on serum biochemistry, tissue weight, voluntary grip strength, maximal aerobic capacity (VO2max), body composition and bone micro-CT and mechanics. RESULTS: Wheel running improved serum biochemistry with decreased creatinine, phosphorous, and parathyroid hormone in the rats with CKD. It improved muscle strength, increased time-to-fatigue (for VO2max), reduced cortical porosity and improved bone microarchitecture. The CKD rats with voluntary wheel access also had reduced kidney cystic weight and reduced left ventricular mass index. CONCLUSIONS: Voluntary wheel running resulted in multiple beneficial systemic effects in rats with CKD and improved their physical function. Studies examining exercise interventions in patients with CKD are warranted.

Loss of Nmp4 optimizes osteogenic metabolism and secretion to enhance bone quality.

A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor nuclear matrix protein 4 (Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared with wild-type (WT) animals. Nmp4-/- mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyperanabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4-/- MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion: a key step in bone anabolism. Nmp4-/- cells showed elevated collagen translation and secretion. The expression of matrix genes that contribute to bone material-level mechanical properties was elevated in Nmp4-/- cells, an observation that was supported by biomechanical testing of bone samples from Nmp4-/- and WT mice. We conclude that loss of Nmp4 increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality.

Substrate Strain Mitigates Effects of ß-Aminopropionitrile-Induced Reduction in Enzymatic Crosslinking.

Enzymatic crosslinks stabilize type I collagen and are catalyzed by lysyl oxidase (LOX), a step interrupted through ß-aminopropionitrile (BAPN) exposure. This study evaluated dose-dependent effects of BAPN on osteoblast gene expression of type I collagen, LOX, and genes associated with crosslink formation. The second objective was to characterize collagen produced in vitro after exposure to BAPN, and to explore changes to collagen properties under continuous cyclical substrate strain. To evaluate dose-dependent effects, osteoblasts were exposed to a range of BAPN dosages (0-10 mM) for gene expression analysis and cell proliferation. Results showed significant upregulation of BMP-1, POST, and COL1A1 and change in cell proliferation. Results also showed that while the gene encoding LOX was unaffected by BAPN treatment, other genes related to LOX activation and matrix production were upregulated. For the loading study, the combined effects of BAPN and mechanical loading were assessed. Gene expression was quantified, atomic force microscopy was used to extract elastic properties of the collagen matrix, and Fourier Transform infrared spectroscopy was used to assess collagen secondary structure for enzymatic crosslinking analysis. BAPN upregulated BMP-1 in static samples and BAPN combined with mechanical loading downregulated LOX when compared to control-static samples. Results showed a higher indentation modulus in BAPN-loaded samples compared to control-loaded samples. Loading increased the mature-to-immature crosslink ratios in control samples, and BAPN increased the height ratio in static samples. In summary, effects of BAPN (upregulation of genes involved in crosslinking, mature/immature crosslinking ratios, upward trend in collagen elasticity) were mitigated by mechanical loading.

Ligand trap of the activin receptor type IIA inhibits osteoclast stimulation of bone remodeling in diabetic mice with chronic kidney disease.

Dysregulation of skeletal remodeling is a component of renal osteodystrophy. Previously, we showed that activin receptor signaling is differentially affected in various tissues in chronic kidney disease (CKD). We tested whether a ligand trap for the activin receptor type 2A (RAP-011) is an effective treatment of the osteodystrophy of the CKD-mineral bone disorder. With a 70% reduction in the glomerular filtration rate, CKD was induced at 14 weeks of age in the ldlr-/- high fat-fed mouse model of atherosclerotic vascular calcification and diabetes. Twenty mice with CKD, hyperphosphatemia, hyperparathyroidism, and elevated activin A were treated with RAP-011, wherease 19 mice were given vehicle twice weekly from week 22 until the mice were killed at 28 weeks of age. The animals were then evaluated by skeletal histomorphometry, micro-computed tomography, mechanical strength testing, and ex vivo bone cell culture. Results in the CKD groups were compared with those of the 16 sham-operated ldlr-/- high fat-fed mice. Sham-operated mice had low-turnover osteodystrophy and skeletal frailty. CKD stimulated bone remodeling with significant increases in osteoclast and osteoblast numbers and bone resorption. Compared with mice with CKD and sham-operated mice, RAP-011 treatment eliminated the CKD-induced increase in these histomorphometric parameters and increased trabecular bone fraction. RAP-011 significantly increased cortical bone area and thickness. Activin A-enhanced osteoclastogenesis was mediated through p-Smad2 association with c-fos and activation of nuclear factor of activated T cells c1 (NFATc1). Thus, an ActRIIA ligand trap reversed CKD-stimulated bone remodeling, likely through inhibition of activin-A induced osteoclastogenesis.

Raloxifene Improves Bone Mechanical Properties in Mice Previously Treated with Zoledronate.

Bisphosphonates represent the gold-standard pharmaceutical agent for reducing fracture risk. Long-term treatment with bisphosphonates can result in tissue brittleness which in rare clinical cases manifests as atypical femoral fracture. Although this has led to an increasing call for bisphosphonate cessation, few studies have investigated therapeutic options for follow-up treatment. The goal of this study was to test the hypothesis that treatment with raloxifene, a drug that has cell-independent effects on bone mechanical material properties, could reverse the compromised mechanical properties that occur following zoledronate treatment. Skeletally mature male C57Bl/6J mice were treated with vehicle (VEH), zoledronate (ZOL), or ZOL followed by raloxifene (RAL; 2 different doses). At the conclusion of 8 weeks of treatment, femora were collected and assessed with microCT and mechanical testing. Trabecular BV/TV was significantly higher in all treated animals compared to VEH with both RAL groups having significantly higher BV/TV compared to ZOL (+21%). All three drug-treated groups had significantly more cortical bone area, higher cortical thickness, and greater moment of inertia at the femoral mid-diaphysis compared to VEH with no difference among the three treated groups. All three drug-treated groups had significantly higher ultimate load compared to VEH-treated animals (+14 to 18%). Both doses of RAL resulted in significantly higher displacement values compared to ZOL-treated animals (+25 to +50%). In conclusion, the current work shows beneficial effects of raloxifene in animals previously treated with zoledronate. The higher mechanical properties of raloxifene-treated animals, combined with similar cortical bone geometry compared to animals treated with zoledronate, suggest that the raloxifene treatment is enhancing mechanical material properties of the tissue.

Raloxifene improves skeletal properties in an animal model of cystic chronic kidney disease.

Patients with chronic kidney disease (CKD) have an increased risk of fracture. Raloxifene is a mild antiresorptive agent that reduces fracture risk in the general population. Here we assessed the impact of raloxifene on the skeletal properties of animals with progressive CKD. Male Cy/+ rats that develop autosomal dominant cystic kidney disease were treated with either vehicle or raloxifene for five weeks. They were assessed for changes in mineral metabolism and skeletal parameters (microCT, histology, whole-bone mechanics, and material properties). Their normal littermates served as controls. Animals with CKD had significantly higher parathyroid hormone levels compared with normal controls, as well as inferior structural and mechanical skeletal properties. Raloxifene treatment resulted in lower bone remodeling rates and higher cancellous bone volume in the rats with CKD. Although it had little effect on cortical bone geometry, it resulted in higher energy to fracture and modulus of toughness values than vehicle-treated rats with CKD, achieving levels equivalent to normal controls. Animals treated with raloxifene had superior tissue-level mechanical properties as assessed by nanoindentation, and higher collagen D-periodic spacing as assessed by atomic force microscopy. Thus, raloxifene can positively impact whole-bone mechanical properties in CKD through its impact on skeletal material properties.

Calcitriol Suppression of Parathyroid Hormone Fails to Improve Skeletal Properties in an Animal Model of Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) leads to complex metabolic changes and an increased risk of fracture. Currently, calcitriol is the standard of care as it effectively suppresses parathyroid hormone (PTH) levels in CKD patients. While calcitriol and its analogs improve BMD and reduce fractures in the general population, the extension of these benefits to patients with advanced kidney disease is unclear. Here, the impact of calcitriol on the skeleton was examined in the setting of reduction in PTH. METHODS: Male Cy/+ rats, a PKD-like CKD model, were treated with either vehicle or calcitriol for 5 weeks. Their normal littermates served as controls. Animals were assessed for changes in mineral metabolism and skeletal parameters (microCT, histology, whole bone mechanics and bone quality). RESULTS: PTH levels were significantly higher (12-fold) in animals with CKD compared to normal controls. CKD animals also exhibited negative changes in bone structural and mechanical properties. Calcitriol treatment resulted in a 60% suppression of PTH levels in animals with CKD. Despite these changes, it had no impact on bone volume (cortical or cancellous), bone turnover, osteoclast number or whole bone mechanical properties. CONCLUSIONS: These data indicate that while calcitriol effectively lowered PTH in rats with CKD, it did little to prevent the negative effects of secondary hyperparathyroidism on the skeleton.

Salubrinal improves mechanical properties of the femur in osteogenesis imperfecta mice.

Salubrinal is an agent that reduces the stress to the endoplasmic reticulum by inhibiting de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). We and others have previously shown that the elevated phosphorylation of eIF2α stimulates bone formation and attenuates bone resorption. In this study, we applied salubrinal to a mouse model of osteogenesis imperfecta (Oim), and examined whether it would improve Oim's mechanical property. We conducted in vitro experiments using RAW264.7 pre-osteoclasts and bone marrow derived cells (BMDCs), and performed in vivo administration of salubrinal to Oim (+/-) mice. The animal study included two control groups (wildtype and Oim placebo). The result revealed that salubrinal decreased expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and suppressed osteoclast maturation, and it stimulated mineralization of mesenchymal stem cells from BMDCs. Furthermore, daily injection of salubrinal at 2 mg/kg for 2 months made stiffness (N/mm) and elastic module (GPa) of the femur undistinguishable to those of the wildtype control. Collectively, this study supported salubrinal's beneficial role to Oim's femora. Unlike bisphosphonates, salubrinal stimulates bone formation. For juvenile OI patients who may favor strengthening bone without inactivating bone remodeling, salubrinal may present a novel therapeutic option.

Large Metal Heads and Vitamin E Polyethylene Increase Frictional Torque in Total Hip Arthroplasty.

PURPOSE: Trunnionosis has reemerged in modern total hip arthroplasty for reasons that remain unclear. Bearing frictional torque transmits forces to the modular head-neck interface, which may contribute to taper corrosion. The purpose of this study is to compare frictional torque of modern bearing couples in total hip arthroplasty. METHODS: Mechanical testing based on in vivo loading conditions was used to measure frictional torque. All bearing couples were lubricated and tested at 1 Hz for more than 2000 cycles. The bearing couples tested included conventional, highly crosslinked (XLPE) and vitamin E polyethylene, CoCr, and ceramic femoral heads and dual-mobility bearings. Statistical analysis was performed using Student t test for single-variable and analysis of variance for multivariant analysis. P ≤ .05 was considered statistically significant. RESULTS: Large CoCr metal heads (≥36 mm) substantially increased frictional torque against XLPE liners (P = .01), a finding not observed in ceramic heads. Vitamin E polyethylene substantially increased frictional torque compared with XLPE in CoCr and ceramic heads (P = .001), whereas a difference between conventional and XLPE was not observed (P = .69) with the numbers available. Dual-mobility bearing with ceramic inner head demonstrated the lowest mean frictional torque of all bearing couples. CONCLUSION: In this simulated in vivo model, large-diameter CoCr femoral heads and vitamin E polyethylene liners are associated with increased frictional torque compared with smaller metal heads and XLPE, respectively. The increased frictional torque of vitamin E polyethylene and larger-diameter femoral heads should be considered and further studied, along with reported benefits of these modern bearing couples.

Effects of fixation and demineralization on bone collagen D-spacing as analyzed by atomic force microscopy.

PURPOSE/AIM: Collagen's role in bone is often considered secondary. As increased attention is paid to collagen, understanding the impact of tissue preservation is important in interpreting experimental results. The goal of this study was to test the hypothesis that bone fixation prior to demineralization would maintain its collagen ultrastructure in an undisturbed state when analyzed using Atomic Force Microscopy (AFM). MATERIALS/METHODS: The anterior diaphysis of a pig femur was cut into 6 mm pieces along its length. Samples were mounted, polished and randomly assigned to control or fixation groups (n = 5/group). Fixation samples were fixed for 24 h prior to demineralization. All samples were briefly demineralized to expose collagen, and imaged using AFM. Mouse tail tendons were also analyzed to explore effects of dehydration and fixation. Measurements from each bone sample were averaged and compared using a Mann-Whitney U-test. Tendon sample means were compared using RMANOVA. To investigate differences in D-spacing distributions, Kolmogorov-Smirnov tests were used. RESULTS: Fixation decreased D-spacing variability within and between bone samples and induced or maintained a higher average D-spacing versus control by shifting the D-spacing population upward. Tendon data indicate that fixing and drying samples leaves collagen near its undisturbed and hydrated native state. DISCUSSION: Fixation in bone prior to demineralization decreased D-spacing variability. D-spacing was shifted upward in fixed samples, indicating that collagen is stretched with mineral present and relaxes upon its removal. The ability to decrease variability in bone suggests that fixation might increase the power to detect changes in collagen due to disease or other pressures.

Multi-scale analysis of bone chemistry, morphology and mechanics in the oim model of osteogenesis imperfecta.

Osteogenesis imperfecta is a congenital disease commonly characterized by brittle bones and caused by mutations in the genes encoding Type I collagen, the single most abundant protein produced by the body. The oim model has a natural collagen mutation, converting its heterotrimeric structure (two α1 and one α2 chains) into α1 homotrimers. This mutation in collagen may impact formation of the mineral, creating a brittle bone phenotype in animals. Femurs from male wild type (WT) and homozygous (oim/oim) mice, all at 12 weeks of age, were assessed using assays at multiple length scales with minimal sample processing to ensure a near-physiological state. Atomic force microscopy (AFM) demonstrated detectable differences in the organization of collagen at the nanoscale that may partially contribute to alterations in material and structural behavior obtained through mechanical testing and reference point indentation (RPI). Changes in geometric and chemical structure obtained from µ-Computed Tomography and Raman spectroscopy indicate a smaller bone with reduced trabecular architecture and altered chemical composition. Decreased tissue material properties in oim/oim mice are likely driven by changes in collagen fibril structure, decreasing space available for mineral nucleation and growth, as supported by a reduction in mineral crystallinity. Multi-scale analyses of this nature offer much in assessing how molecular changes compound to create a degraded, brittle bone phenotype.

Compromised femoral and lumbovertebral bone in the Dp(16)1Yey Down syndrome mouse model.

Down syndrome (DS), affecting ∼1 in 800 live births, is caused by the triplication of human chromosome 21 (Hsa21). Individuals with DS have skeletal features including craniofacial abnormalities and decreased bone mineral density (BMD). Lowered BMD can lead to increased fracture risk, with common fracture points at the femoral neck and lumbar spine. While the femur has been studied in DS mouse models, there is little research done on the vertebrae despite evidence that humans with DS have affected vertebrae. Additionally, it is important to establish when skeletal deficits occur to find times of potential intervention. The Dp(16)1Yey DS mouse model has all genes triplicated on mouse chromosome 16 orthologous to Hsa21 and displayed deficits in long bone, including trabecular and cortical deficits in male but not female mice, at 12 weeks. We hypothesized that the long bone and lumbovertebral microarchitecture would exhibit sexually dimorphic deficits in Dp(16)1Yey mice compared to control mice and long bone strength would be diminished in Dp(16)1Yey mice at 6 weeks. The trabecular region of the 4th lumbar (L4) vertebra and the trabecular and cortical regions of the femur were analyzed via micro-computed tomography and 3-point bending in 6-week-old male and female Dp(16)1Yey and control mice. Trabecular and cortical deficits were observed in femurs from male Dp(16)1Yey mice, and cortical deficits were seen in femurs of male and female Dp(16)1Yey mice. Male Dp(16)1Yey femurs had more deficits in bone strength at whole bone and tissue-estimate level properties, but female Dp(16)1Yey mice were also affected. Additionally, the L4 of male and female Dp(16)1Yey mice show trabecular deficits, which have not been previously reported in a DS mouse model. Our results indicate that skeletal deficits associated with DS occur early in skeletal development, are dependent on skeletal compartment and site, are sex dependent, and potential interventions should likely begin early in skeletal development of DS mouse models.

Combining anabolic loading and raloxifene improves bone quantity and some quality measures in a mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) increases fracture risk due to changes in bone quantity and quality caused by mutations in collagen and its processing proteins. Current therapeutics improve bone quantity, but do not treat the underlying quality deficiencies. Male and female G610C+/- mice, a murine model of OI, were treated with a combination of raloxifene and in vivo axial tibial compressive loading starting at 10 weeks of age and continuing for 6 weeks to improve bone quantity and quality. Bone geometry and mechanical properties were measured to determine whole bone and tissue-level material properties. A colocalized Raman/nanoindentation system was used to measure chemical composition and nanomechanical properties in newly formed bone compared to old bone to determine if bone formed during the treatment regimen differed in quality compared to bone formed prior to treatment. Lastly, lacunar geometry and osteocyte apoptosis were assessed. OI mice were able to build bone in response to the loading, but this response was less robust than in control mice. Raloxifene improved some bone material properties in female but not male OI mice. Raloxifene did not alter nanomechanical properties, but loading did. Lacunar geometry was largely unchanged with raloxifene and loading. However, osteocyte apoptosis was increased with loading in raloxifene treated female mice. Overall, combination treatment with raloxifene and loading resulted in positive but subtle changes to bone quality.