Water constitutes roughly a quarter of the cortical bone by volume yet can greatly influence mechanical properties and tissue quality. There is a growing appreciation for how water can dynamically ...change due to age, disease, and treatment. A key emerging area related to bone mechanical and tissue properties lies in differentiating the role of water in its four different compartments, including free/pore water, water loosely bound at the collagen/mineral interfaces, water tightly bound within collagen triple helices, and structural water within the mineral. This review summarizes our current knowledge of bone water across the four functional compartments and discusses how alterations in each compartment relate to mechanical changes. It provides an overview on the advent of- and improvements to- imaging and spectroscopic techniques able to probe nano-and molecular scales of bone water. These technical advances have led to an emerging understanding of how bone water changes in various conditions, of which aging, chronic kidney disease, diabetes, osteoporosis, and osteogenesis imperfecta are reviewed. Finally, it summarizes work focused on therapeutically targeting water to improve mechanical properties.
•Bone water can greatly influence mechanical properties and tissue quality.•Bone water is found as free/pore, loosely bound, tightly bound, and structural water.•Bone hydration can dynamically change due to healthy aging, disease, and treatment.•Imaging and spectroscopic advances have made the study of bone water possible.•Therapeutically targeting bone water may be efficacious in improving mechanical properties.
Thermoregulation is an important factor that could have physiological consequences on pre-clinical research outcomes. Simply housing mice at thermoneutral temperature has been shown to prevent the ...well-established loss of cancellous bone that is typical in growing mice. In this study, active tissue formation was induced by non-invasive tibial loading in female mice and combined with raloxifene treatment to assess whether temperature could enhance their combined effects on bone morphology and mechanical properties. It was hypothesized that by removing the cold stress under which normal lab mice are housed, a metabolic boost would allow for further architectural and mechanical improvements in mice exposed to a combination of tibial loading and raloxifene. Ten-week old female C57BL/6J mice were treated with raloxifene, underwent tibial loading to a maximum tensile stress of 2050 με, and were housed in thermoneutral conditions (32 °C) for 6 weeks. We investigated bone morphology through microcomputed tomography (μCT), mechanical properties via four-point bending, and fracture toughness testing. Results confirmed previous work showing a combined effect of external loading and raloxifene which led to greater improvements in most properties than either individual treatment. Counter to the hypothesis, temperature had modest effects on body weight, overall bone size, and trabecular architecture, and most effects were detrimental. Thermoneutrality had no impact on mechanical integrity or fracture toughness. In most cases, the magnitude of temperature-based effects were less robust than either RAL treatment or loading.
•Thermal physiological characteristics of mice can impact research outcomes.•Thermoneutrality was superimposed on tibial compression and Raloxifene treatment.•Temperature modestly impacted weight and bone architecture, often detrimentally.•Thermoneutrality had no impact on mechanical integrity.•Raloxifene and loading resulted in additive improvements in bone properties.
Abstract Diabetes detrimentally affects the musculoskeletal system by stiffening the collagen matrix due to increased advanced glycation end products (AGEs). In this study, tibiae and tendon from ...Zucker diabetic Sprague–Dawley (ZDSD) rats were compared to Sprague–Dawley derived controls (CD) using Atomic Force Microscopy. ZDSD and CD tibiae were compared using Raman Spectroscopy and Reference Point Indentation (RPI). ZDSD bone had a significantly different distribution of collagen D-spacing than CD (p = 0.015; ZDSD n = 294 fibrils; CD n = 274 fibrils) which was more variable and shifted to higher values. This shift between ZDSD and CD D-spacing distribution was more pronounced in tendon (p < 0.001; ZDSD n = 350; CD n = 371). Raman revealed significant increases in measures of bone matrix mineralization in ZDSD (PO4 3 − ν1/Amide I p = 0.008; PO4 3 − ν1/CH2 wag p = 0.047; n = 5 per group) despite lower bone mineral density (aBMD) and ash fraction indicating diabetes may preferentially reduce the Raman signature of collagen. Decreased indentation distance increase (p = 0.010) and creep indentation distance (p = 0.040) measured by RPI (n = 9 per group) in ZDSD rats suggest a matrix more resistant to indentation under the high stresses associated with RPI at this length scale. There were significant correlations between Raman and RPI measurements in the ZDSD population (n = 18 locations) but not the CD population (n = 16 locations) indicating that while RPI is relatively unaffected by biological noise, it is sensitive to disease-induced compositional changes. In conclusion, diabetes in the ZDSD rat causes changes to the nanoscale morphology of collagen that result in compositional and mechanical effects in bone at the microscale.
OBJECTIVE The Barrow Ruptured Aneurysm Trial (BRAT) is a prospective, randomized trial in which treatment with clipping was compared to treatment with coil embolization. Patients were randomized to ...treatment on presentation with any nontraumatic subarachnoid hemorrhage (SAH). Because all other randomized trials comparing these 2 types of treatments have been limited to saccular aneurysms, the authors analyzed the current BRAT data for this subgroup of lesions. METHODS The primary BRAT analysis included all sources of SAH: nonaneurysmal lesions; saccular, blister, fusiform, and dissecting aneurysms; and SAHs from an aneurysm associated with either an arteriovenous malformation or a fistula. In this post hoc review, the outcomes for the subgroup of patients with saccular aneurysms were further analyzed by type of treatment. The extent of aneurysm obliteration was adjudicated by an independent neuroradiologist not involved in treatment. RESULTS Of the 471 patients enrolled in the BRAT, 362 (77%) had an SAH from a saccular aneurysm. Patients with saccular aneurysms were assigned equally to the clipping and the coiling cohorts (181 each). In each cohort, 3 patients died before treatment and 178 were treated. Of the 178 clip-assigned patients with saccular aneurysms, 1 (1%) was crossed over to coiling, and 64 (36%) of the 178 coil-assigned patients were crossed over to clipping. There was no statistically significant difference in poor outcome (modified Rankin Scale score > 2) between these 2 treatment arms at any recorded time point during 6 years of follow-up. After the initial hospitalization, 1 of 241 (0.4%) clipped saccular aneurysms and 21 of 115 (18%) coiled saccular aneurysms required retreatment (p < 0.001). At the 6-year follow-up, 95% (95/100) of the clipped aneurysms were completely obliterated, compared with 40% (16/40) of the coiled aneurysms (p < 0.001). There was no difference in morbidity between the 2 treatment groups (p = 0.10). CONCLUSIONS In the subgroup of patients with saccular aneurysms enrolled in the BRAT, there was no significant difference between modified Rankin Scale outcomes at any follow-up time in patients with saccular aneurysms assigned to clipping compared with those assigned to coiling (intent-to-treat analysis). At the 6-year follow-up evaluation, rates of retreatment and complete aneurysm obliteration significantly favored patients who underwent clipping compared with those who underwent coiling. Clinical trial registration no.: NCT01593267 (clinicaltrials.gov).
Chronic kidney disease (CKD), which leads tocortical bone loss and increasedporosity,increases therisk of fracture. Animal models have confirmed that these changes compromise whole bone mechanical ...properties. Estimates from whole bone testing suggest that material properties are negatively affected, though tissue-level assessmentshavenot been conducted. Therefore, the goal of the present study was to examine changes in cortical bone at different length scales using a rat model with theprogressive development of CKD. At 30 weeks of age (∼75% reduction in kidney function), skeletally mature male Cy/+ rats were compared to their normal littermates. Cortical bone material propertieswere assessed with reference point indentation (RPI), atomic force microscopy (AFM), Raman spectroscopy,and high performance liquid chromatography (HPLC). Bones from animals with CKD had higher (+18%) indentation distance increase and first cycle energy dissipation (+8%) as measured by RPI.AFM indentation revealed a broader distribution of elastic modulus values in CKD animals witha greater proportion of both higher and lower modulus values compared to normal controls. Yet, tissue composition, collagen morphology, and collagen cross-linking fail to account for these differences. Though the specific skeletal tissue alterations responsible for these mechanical differences remain unclear, these results indicate that cortical bone material properties are altered in these animals and may contribute to the increased fracture risk associated with CKD.
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.
Computation has guided the design of conformationally-strained dioxolane-fused
-cyclooctene (d-TCO) derivatives that display excellent reactivity in the tetrazine ligation. A water soluble derivative ...of 3,6-dipyridyl-
-tetrazine reacts with d-TCO with a second order rate
366,000 (+/- 15,000) M
s
at 25 °C in pure water. Furthermore, d-TCO derivatives can be prepared easily, are accessed through diastereoselective synthesis, and are typically crystalline bench-stable solids that are stable in aqueous solution, blood serum, or in the presence of thiols in buffered solution. GFP with a genetically encoded tetrazine-containing amino acid was site-specifically labelled
by a d-TCO derivative. The fastest bioorthogonal reaction reported to date
3,300,000 (+/- 40,000) M
s
in H
O at 25 °C is described herein with a cyclopropane-fused
-cyclooctene. d-TCO derivatives display rates within an order of magnitude of these fastest
-cyclooctene reagents, and also display enhanced stability and aqueous solubility.
Microphthalmia-associated transcription factor (MITF) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. ...In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A, -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model system.
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.
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.
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.
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