Bone Fragility in High Fat Diet-induced Obesity is Partially Independent of Type 2 Diabetes in Mice.

Obesity and type 2 diabetes (T2D) are risk factors for fragility fractures. It is unknown whether this elevated risk is due to a diet favoring obesity or the diabetes that often occurs with obesity. Therefore, we hypothesized that the fracture resistance of bone is lower in mice fed with a high fat diet (45% kcal; HFD) than in mice that fed on a similar, control diet (10% kcal; LFD), regardless of whether the mice developed overt T2D. Sixteen-week-old, male NON/ShiLtJ mice (resistant to T2D) and age-matched, male NONcNZO10/LtJ (prone to T2D) received a control LFD or HFD for 21 weeks. HFD increased the bodyweight to a greater extent in the ShiLtJ mice compared to the NZO10 mice, while blood glucose levels were significantly higher in NZO10 than in ShiLtJ mice. As such, the glycated hemoglobin A1c (HbA1c) levels exceeded 10% in NZO10 mice, but it remained below 6% in ShiLtJ mice. Diet did not affect HbA1c. HFD lowered trabecular number and bone volume fraction of the distal femur metaphysis (micro-computed tomography or µCT) in both strains. For the femur mid-diaphysis, HFD significantly reduced the yield moment (mechanical testing by three-point bending) in both strains but did not affect cross-sectional bone area, cortical thickness, nor cortical tissue mineral density (µCT). Furthermore, the effect of diet on yield moment was independent of the structural resistance of the femur mid-diaphysis suggesting a negative effect of HFD on characteristics of the bone matrix. However, neither Raman spectroscopy nor assays of advanced glycation end-products identified how HFD affected the matrix. HFD also lowered the resistance of cortical bone to crack growth in only the diabetic NZO10 mice (fracture toughness testing of other femur), while HFD reduced the ultimate force of the L6 vertebra in both strains (compression testing). In conclusion, the HFD-related decrease in bone strength can occur in mice resistant and prone to diabetes indicating that a diet high in fat deleteriously affects bone without necessarily causing hyperglycemia.

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.

The Utility of AI in Writing a Scientific Review Article on the Impacts of COVID-19 on Musculoskeletal Health.

PURPOSE OF REVIEW: There were two primary purposes to our reviews. First, to provide an update to the scientific community about the impacts of COVID-19 on musculoskeletal health. Second, was to determine the value of using a large language model, ChatGPT 4.0, in the process of writing a scientific review article. To accomplish these objectives, we originally set out to write three review articles on the topic using different methods to produce the initial drafts of the review articles. The first review article was written in the traditional manner by humans, the second was to be written exclusively using ChatGPT (AI-only or AIO), and the third approach was to input the outline and references selected by humans from approach 1 into ChatGPT, using the AI to assist in completing the writing (AI-assisted or AIA). All review articles were extensively fact-checked and edited by all co-authors leading to the final drafts of the manuscripts, which were significantly different from the initial drafts. RECENT FINDINGS: Unfortunately, during this process, it became clear that approach 2 was not feasible for a very recent topic like COVID-19 as at the time, ChatGPT 4.0 had a cutoff date of September 2021 and all articles published after this date had to be provided to ChatGPT, making approaches 2 and 3 virtually identical. Therefore, only two approaches and two review articles were written (human and AI-assisted). Here we found that the human-only approach took less time to complete than the AI-assisted approach. This was largely due to the number of hours required to fact-check and edit the AI-assisted manuscript. Of note, the AI-assisted approach resulted in inaccurate attributions of references (about 20%) and had a higher similarity index suggesting an increased risk of plagiarism. The main aim of this project was to determine whether the use of AI could improve the process of writing a scientific review article. Based on our experience, with the current state of technology, it would not be advised to solely use AI to write a scientific review article, especially on a recent topic.

COVID-19 and Bone Loss: A Review of Risk Factors, Mechanisms, and Future Directions.

PURPOSE OF REVIEW: SARS-CoV-2 drove the catastrophic global phenomenon of the COVID-19 pandemic resulting in a multitude of systemic health issues, including bone loss. The purpose of this review is to summarize recent findings related to bone loss and potential mechanisms. RECENT FINDINGS: The early clinical evidence indicates an increase in vertebral fractures, hypocalcemia, vitamin D deficiencies, and a loss in BMD among COVID-19 patients. Additionally, lower BMD is associated with more severe SARS-CoV-2 infection. Preclinical models have shown bone loss and increased osteoclastogenesis. The bone loss associated with SARS-CoV-2 infection could be the result of many factors that directly affect the bone such as higher inflammation, activation of the NLRP3 inflammasome, recruitment of Th17 cells, the hypoxic environment, and changes in RANKL/OPG signaling. Additionally, SARS-CoV-2 infection can exert indirect effects on the skeleton, as mechanical unloading may occur with severe disease (e.g., bed rest) or with BMI loss and muscle wasting that has also been shown to occur with SARS-CoV-2 infection. Muscle wasting can also cause systemic issues that may influence the bone. Medications used to treat SARS-CoV-2 infection also have a negative effect on the bone. Lastly, SARS-CoV-2 infection may also worsen conditions such as diabetes and negatively affect kidney function, all of which could contribute to bone loss and increased fracture risk. SARS-CoV-2 can negatively affect the bone through multiple direct and indirect mechanisms. Future work will be needed to determine what patient populations are at risk of COVID-19-related increases in fracture risk, the mechanisms behind bone loss, and therapeutic options. This review article is part of a series of multiple manuscripts designed to determine the utility of using artificial intelligence for writing scientific reviews.

SARS-CoV-2 and its Multifaceted Impact on Bone Health: Mechanisms and Clinical Evidence.

PURPOSE OF REVIEW: SARS-CoV-2 infection, the culprit of the COVID-19 pandemic, has been associated with significant long-term effects on various organ systems, including bone health. This review explores the current understanding of the impacts of SARS-CoV-2 infection on bone health and its potential long-term consequences. RECENT FINDINGS: As part of the post-acute sequelae of SARS-CoV-2 infection, bone health changes are affected by COVID-19 both directly and indirectly, with multiple potential mechanisms and risk factors involved. In vitro and preclinical studies suggest that SARS-CoV-2 may directly infect bone marrow cells, leading to alterations in bone structure and osteoclast numbers. The virus can also trigger a robust inflammatory response, often referred to as a "cytokine storm", which can stimulate osteoclast activity and contribute to bone loss. Clinical evidence suggests that SARS-CoV-2 may lead to hypocalcemia, altered bone turnover markers, and a high prevalence of vertebral fractures. Furthermore, disease severity has been correlated with a decrease in bone mineral density. Indirect effects of SARS-CoV-2 on bone health, mediated through muscle weakness, mechanical unloading, nutritional deficiencies, and corticosteroid use, also contribute to the long-term consequences. The interplay of concurrent conditions such as diabetes, obesity, and kidney dysfunction with SARS-CoV-2 infection further complicates the disease's impact on bone health. SARS-CoV-2 infection directly and indirectly affects bone health, leading to potential long-term consequences. This review article is part of a series of multiple manuscripts designed to determine the utility of using artificial intelligence for writing scientific reviews.

Effects of novel raloxifene analogs alone or in combination with mechanical loading in the Col1a2G610c/+ murine model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a hereditary bone disease in which gene mutations affect collagen formation, leading to a weak, brittle bone phenotype that can cause severe skeletal deformity and increased fracture risk. OI interventions typically repurpose osteoporosis medications to increase bone mass, but this approach does not address compromised tissue-level material properties. Raloxifene (RAL) is a mild anti-resorptive used to treat osteoporosis that has also been shown to increase bone strength by a-cellularly increasing bone bound water content, but RAL cannot be administered to children due to its hormonal activity. The goal of this study was to test a RAL analog with no estrogen receptor (ER) signaling but maintained ability to reduce fracture risk. The best performing analog from a previous analog characterization project, named RAL-ADM, was tested in an in vivo study. Female wildtype (WT) and Col1a2G610C/+ (G610C) mice were randomly assigned to treated or untreated groups, for a total of 4 groups (n = 15). Starting at 10 weeks of age, all mice underwent compressive tibial loading 3×/week to induce an anabolic bone formation response in conjunction with RAL-ADM treatment (0.5 mg/kg; 5×/week) for 6 weeks. Tibiae were scanned via microcomputed tomography then tested to failure in four-point bending. RAL-ADM had reduced ER affinity, and increased post-yield properties, but did not improve bone strength in OI animals, suggesting some properties can be improved by RAL analogs but further development is needed to create an analog with decidedly positive impacts to OI bone.

Fragile X Messenger Ribonucleoprotein 1 (FMR1), a novel inhibitor of osteoblast/osteocyte differentiation, regulates bone formation, mass, and strength in young and aged male and female mice.

Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene mutations lead to fragile X syndrome, cognitive disorders, and, in some individuals, scoliosis and craniofacial abnormalities. Four-month-old (mo) male mice with deletion of the FMR1 gene exhibit a mild increase in cortical and cancellous femoral bone mass. However, consequences of absence of FMR1 in bone of young/aged male/female mice and the cellular basis of the skeletal phenotype remain unknown. We found that absence of FMR1 results in improved bone properties with higher bone mineral density in both sexes and in 2- and 9-mo mice. The cancellous bone mass is higher only in females, whereas, cortical bone mass is higher in 2- and 9-mo males, but higher in 2- and lower in 9-mo female FMR1-knockout mice. Furthermore, male bones show higher biomechanical properties at 2mo, and females at both ages. Absence of FMR1 increases osteoblast/mineralization/bone formation and osteocyte dendricity/gene expression in vivo/ex vivo/in vitro, without affecting osteoclasts in vivo/ex vivo. Thus, FMR1 is a novel osteoblast/osteocyte differentiation inhibitor, and its absence leads to age-, site- and sex-dependent higher bone mass/strength.

Correlation analysis of cartilage wear with biochemical composition, viscoelastic properties and friction.

Healthy articular cartilage exhibits remarkable resistance to wear, sustaining mechanical loads and relative motion for decades. However, tissues that replace or repair cartilage defects are much less long lasting. Better information on the compositional and material characteristics that contribute to the wear resistance of healthy cartilage could help guide strategies to replace and repair degenerated tissue. The main objective of this study was to assess the relationship between wear of healthy articular cartilage, its biochemical composition, and its viscoelastic material properties. The correlation of these factors with the coefficient of friction during the wear test was also evaluated. Viscoelastic properties of healthy bovine cartilage were determined via stress relaxation indentation. The same specimens underwent an accelerated, in vitro wear test, and the amount of glycosaminoglycans (GAGs) and collagen released during the wear test were considered measures of wear. The frictional response during the wear test was also recorded. The GAG, collagen and water content and the concentration of the enzymatic collagen crosslink pyridinoline were quantified in tissue that was adjacent to each wear test specimen. Finally, correlation analysis was performed to identify potential relationships between wear characteristics of healthy articular cartilage with its composition, viscoelastic material properties and friction. The findings suggest that stiffer cartilage with higher GAG, collagen and water content has a higher wear resistance. Enzymatic collagen crosslinks also enhance the wear resistance of the collagen network. The parameters of wear, composition, and mechanical stiffness of cartilage were all correlated with one another, suggesting that they are interrelated. However, friction was largely independent of these in this study. The results identify characteristics of healthy articular cartilage that contribute to its remarkable wear resistance. These data may be useful for guiding techniques to restore, regenerate, and stabilize cartilage tissue.

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.

Dietary supplements do not improve bone morphology or mechanical properties in young female C57BL/6 mice.

Bone is a hierarchical material formed by an organic extracellular matrix and mineral where each component and their physical relationship with each other contribute to fracture resistance. Bone quality can be affected by nutrition, and dietary supplements that are marketed to improve overall health may improve the fracture resistance of bone. To test this, 11 week old female C57BL/6 mice were fed either collagen, chondroitin sulfate, glucosamine sulfate, or fish oil 5 times a week for 8 weeks. Femurs, tibiae, and vertebrae were scanned with micro-computed tomography and then mechanically tested. Glucosamine and fish oil lowered elastic modulus, but did not alter the overall strength of the femur. There were no differences in bone mechanics of the tibiae or vertebrae. Overall, the data suggest that dietary supplements did little to improve bone quality in young, healthy mice. These supplements may be more effective in diseased or aged mice.

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.

Morphological and mechanical characterization of bone phenotypes in the Amish G610C murine model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a hereditary bone disease where gene mutations affect Type I collagen formation resulting in osteopenia and increased fracture risk. There are several established mouse models of OI, but some are severe and result in spontaneous fractures or early animal death. The Amish Col1a2G610C/+ (G610C) mouse model is a newer, moderate OI model that is currently being used in a variety of intervention studies, with differing background strains, sexes, ages, and bone endpoints. This study is a comprehensive mechanical and architectural characterization of bone in G610C mice bred on a C57BL/6 inbred strain and will provide a baseline for future treatment studies. Male and female wild-type (WT) and G610C mice were euthanized at 10 and 16 weeks (n = 13-16). Harvested tibiae, femora, and L4 vertebrae were scanned via micro-computed tomography and analyzed for cortical and trabecular architectural properties. Femora and tibiae were then mechanically tested to failure. G610C mice had less bone but more highly mineralized cortical and trabecular tissue than their sex- and age-matched WT counterparts, with cortical cross-sectional area, thickness, and mineral density, and trabecular bone volume, mineral density, spacing, and number all differing significantly as a function of genotype (2 Way ANOVA with main effects of sex and genotype at each age). In addition, mechanical yield force, ultimate force, displacement, strain, and toughness were all significantly lower in G610C vs. WT, highlighting a brittle phenotype. This characterization demonstrates that despite being a moderate OI model, the Amish G610C mouse model maintains a distinctly brittle phenotype and is well-suited for use in future intervention studies.

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.

Post-translational modifications in collagen type I of bone in a mouse model of aging.

The fracture resistance of cortical bone and matrix hydration are known to decline with advanced aging. However, the underlying mechanisms remain poorly understood, and so we investigated levels of matrix proteins and post-translational modifications (PTM) of collagen I in extracts from the tibia of 6-mo. and 20-mo. old BALB/c mice (female and male analysis done separately). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the levels of collagen I deamidation at specific asparagine (Asn) and glutamine (Gln) residues significantly increased with age. Other non-enzymatic PTMs such as carboxymethylation of lysine (CML) were detected as well, but the relative abundance did not vary with age. No significant age-related differences in the abundance of hydroxylysine glycosylation sites were found, but hydroxylation levels at a few of the numerous lysine and proline hydroxylation sites significantly changed by a small amount with age. We performed molecular modeling and dynamics (MD) simulations for three triple helical fragments representing collagen I regions with prominent age-dependent increases in deamidation as identified by LC-MS/MS of male extracts. These 3 fragments included deamidated Asn and Gln residues as follows: 1) an Asn428 site of the α2(I) chain in which deamidation levels increased from 4.4% at 6-mo. to 8.1% at 20-mo., 2) an Asn983 site of the α2(I) chain with a deamidation increase from 18.3% to 36.8% with age and an Asn1052 site of the α1(I) chain with consistent deamidation levels of ~60% across the age groups, and 3) a Gln410 site of the α1(I) chain that went from no detectable deamidation at 6-mo. to 2.7% at 20-mo. and a neighboring Asn421 site of the same chain with an age-related deamidation increase from 3.6% to 16.3%. The deamidation levels at these sites inversely correlated with an estimate of toughness determined from three-point bending tests of the femur mid-diaphysis. MD revealed that the sidechains become more negatively charged at deamidated sites and that deamidation alters hydrogen bonding with water along the collagen backbone while increasing water interactions with the aspartic and glutamic acid sidechains. Our findings suggest a new mechanism of the age-dependent reduction in the fracture resistance of cortical bone whereby deamidation of Asn and Glu residues redistributes bound water within collagen I triple helix.

The BALB/c mouse as a preclinical model of the age-related deterioration in the lumbar vertebra.

The likelihood of experiencing an osteoporotic fracture of one or more vertebral bodies increases with age, and this increase is not solely due to sex steroid deficiency. For the purpose of assessing the effectiveness of novel therapeutic strategies in the prevention of vertebral fractures among the elderly, we hypothesized that the BALB/c mouse model of aging phenocopies the age-related decrease in human VB strength. To test this hypothesis, we assessed the age-related changes in trabecular architecture of the L6 VB, with respect to those in the distal femur metaphysis, between 6-mo. (young adulthood, n = 20/sex) and 20-mo. of age (old age, n = 18/sex) and then determined how well the architectural characteristics, volumetric bone mineral density (vBMD), and predicted failure force from µCT-derived finite element analysis (µFEA) with linear elastic failure criteria explained the age-related variance in VB strength, which was the ultimate force during quasi-static loading of the VB in compression. While there was a pronounced age-related deterioration in trabecular architecture in the distal femur metaphysis of female and male BALB/c mice, the decrease in trabecular bone volume fraction and trabecular number between 6-mo. and 20-mo. of age occurred in male mice, but not in female mice. As such, the VB strength was lower with age in males only. Nonetheless, BV/TV and volumetric bone mineral density (vBMD) positively correlated with the ultimate compressive force of the L6 VB for both females and males. Whether using a fixed homogeneous distribution of tissue modulus (Et = 18 GPa) or a heterogeneous distribution of Et based on a positive relationship with TMD, the predicted failure force of the VB was not independent of age, thereby suggesting linear µFEA may not be a suitable replacement for mechanical-based measurements of strength with respect to age-related changes. Overall, the BALB/c mouse model of aging mimics the age-related in decline in human VB strength when comparing 6-mo. and 20-mo. old male mice. The decrease in VB strength in female mice may occur over a different age range.

Control of Bone Matrix Properties by Osteocytes.

Osteocytes make up 90-95% of the cellular content of bone and form a rich dendritic network with a vastly greater surface area than either osteoblasts or osteoclasts. Osteocytes are well positioned to play a role in bone homeostasis by interacting directly with the matrix; however, the ability for these cells to modify bone matrix remains incompletely understood. With techniques for examining the nano- and microstructure of bone matrix components including hydroxyapatite and type I collagen becoming more widespread, there is great potential to uncover novel roles for the osteocyte in maintaining bone quality. In this review, we begin with an overview of osteocyte biology and the lacunar-canalicular system. Next, we describe recent findings from in vitro models of osteocytes, focusing on the transitions in cellular phenotype as they mature. Finally, we describe historical and current research on matrix alteration by osteocytes in vivo, focusing on the exciting potential for osteocytes to directly form, degrade, and modify the mineral and collagen in their surrounding matrix.

The age-related decrease in material properties of BALB/c mouse long bones involves alterations to the extracellular matrix.

One possibility for the disproportionate increase in fracture risk with aging relative to the decrease in bone mass is an accumulation of changes to the bone matrix which deleteriously affect fracture resistance. In order to effectively develop new targets for osteoporosis, a preclinical model of the age-related loss in fracture resistance needs to be established beyond known age-related decreases in bone mineral density and bone volume fraction. To that end, we examined long bones of male and female BALB/c mice at 6-mo. and 20-mo. of age and assessed whether material and matrix properties of cortical bone significantly differed between the age groups. The second moment of area of the diaphysis (minimum and maximum principals for femur and radius, respectively) as measured by ex vivo micro-computed tomography (µCT) was higher at 20-mo. than at 6-mo. for both males and females, but ultimate moment as measured by three-point bending tests did not decrease with age. Cortical thickness was lower with age for males, but higher for old females. Partially accounting for differences in structure, material estimates of yield, ultimate stress, and toughness (left femur) were 12.6%, 11.1%, and 40.9% lower, respectively, with age for both sexes. The ability of the cortical bone to resist crack growth (right femur) was also 18.1% less for the old than for the young adult mice. These decreases in material properties were not due to changes in intracortical porosity as pore number decreased with age. Rather, age-related alterations in the matrix were observed for both sexes: enzymatic and non-enzymatic crosslinks by high performance liquid chromatography increased (femur), volume fraction of bound water by 1H-nuclear magnetic resonance relaxometry decreased (femur), cortical tissue mineral density by µCT increased (femur and radius), and an Amide I sub-peak ratio I1670/I1640 by Raman spectroscopy increased (tibia). Overall, there are multiple matrix changes to potentially target that could prevent the age-related decrease in fracture resistance observed in BALB/c mouse.

Effect of Advanced Glycation End-Products (AGE) Lowering Drug ALT-711 on Biochemical, Vascular, and Bone Parameters in a Rat Model of CKD-MBD.

Chronic kidney disease-mineral bone disorder (CKD-MBD) is a systemic disorder that affects blood measures of bone and mineral homeostasis, vascular calcification, and bone. We hypothesized that the accumulation of advanced glycation end-products (AGEs) in CKD may be responsible for the vascular and bone pathologies via alteration of collagen. We treated a naturally occurring model of CKD-MBD, the Cy/+ rat, with a normal and high dose of the AGE crosslink breaker alagebrium (ALT-711), or with calcium in the drinking water to mimic calcium phosphate binders for 10 weeks. These animals were compared to normal (NL) untreated animals. The results showed that CKD animals, compared to normal animals, had elevated blood urea nitrogen (BUN), PTH, FGF23 and phosphorus. Treatment with ALT-711 had no effect on kidney function or PTH, but 3 mg/kg lowered FGF23 whereas calcium lowered PTH. Vascular calcification of the aorta assessed biochemically was increased in CKD animals compared to NL, and decreased by the normal, but not high dose of ALT-711, with parallel decreases in left ventricular hypertrophy. ALT-711 (3 mg/kg) did not alter aorta AGE content, but reduced aorta expression of receptor for advanced glycation end products (RAGE) and NADPH oxidase 2 (NOX2), suggesting effects related to decreased oxidative stress at the cellular level. The elevated total bone AGE was decreased by 3 mg/kg ALT-711 and both bone AGE and cortical porosity were decreased by calcium treatment, but only calcium improved bone properties. In summary, treatment of CKD-MBD with an AGE breaker ALT-711, decreased FGF23, reduced aorta calcification, and reduced total bone AGE without improvement of bone mechanics. These results suggest little effect of ALT-711 on collagen, but potential cellular effects. The data also highlights the need to better measure specific types of AGE proteins at the tissue level in order to fully elucidate the impact of AGEs on CKD-MBD. © 2019 American Society for Bone and Mineral Research.

PD-1 up-regulation on CD4+ T cells promotes pulmonary fibrosis through STAT3-mediated IL-17A and TGF-ß1 production.

Pulmonary fibrosis is a progressive inflammatory disease with high mortality and limited therapeutic options. Previous genetic and immunologic investigations suggest common intersections between idiopathic pulmonary fibrosis (IPF), sarcoidosis, and murine models of pulmonary fibrosis. To identify immune responses that precede collagen deposition, we conducted molecular, immunohistochemical, and flow cytometric analysis of human and murine specimens. Immunohistochemistry revealed programmed cell death-1 (PD-1) up-regulation on IPF lymphocytes. PD-1+CD4+ T cells with reduced proliferative capacity and increased transforming growth factor-ß (TGF-ß)/interleukin-17A (IL-17A) expression were detected in IPF, sarcoidosis, and bleomycin CD4+ T cells. PD-1+ T helper 17 cells are the predominant CD4+ T cell subset expressing TGF-ß. Coculture of PD-1+CD4+ T cells with human lung fibroblasts induced collagen-1 production. Strikingly, ex vivo PD-1 pathway blockade resulted in reductions in TGF-ß and IL-17A expression from CD4+ T cells, with concomitant declines in collagen-1 production from fibroblasts. Molecular analysis demonstrated PD-1 regulation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Chemical blockade of STAT3, using the inhibitor STATTIC, inhibited collagen-1 production. Both bleomycin administration to PD-1 null mice or use of antibody against programmed cell death ligand 1 (PD-L1) demonstrated significantly reduced fibrosis compared to controls. This work identifies a critical, previously unrecognized role for PD-1+CD4+ T cells in pulmonary fibrosis, supporting the use of readily available therapeutics that directly address interstitial lung disease pathophysiology.

The Role of Matrix Composition in the Mechanical Behavior of Bone.

PURPOSE OF REVIEW: While thinning of the cortices or trabeculae weakens bone, age-related changes in matrix composition also lower fracture resistance. This review summarizes how the organic matrix, mineral phase, and water compartments influence the mechanical behavior of bone, thereby identifying characteristics important to fracture risk. RECENT FINDINGS: In the synthesis of the organic matrix, tropocollagen experiences various post-translational modifications that facilitate a highly organized fibril of collagen I with a preferred orientation giving bone extensibility and several toughening mechanisms. Being a ceramic, mineral is brittle but increases the strength of bone as its content within the organic matrix increases. With time, hydroxyapatite-like crystals experience carbonate substitutions, the consequence of which remains to be understood. Water participates in hydrogen bonding with organic matrix and in electrostatic attractions with mineral phase, thereby providing stability to collagen-mineral interface and ductility to bone. Clinical tools sensitive to age- and disease-related changes in matrix composition that the affect mechanical behavior of bone could potentially improve fracture risk assessment.