The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation

Mineralization is a ubiquitous process in the animal kingdom and is fundamental to human development and health. Dysfunctional or aberrant mineralization leads to a variety of medical problems, and so an understanding of these processes is essential to their mitigation. Osteoblasts create the nano-composite structure of bone by secreting a collagenous extracellular matrix (ECM) on which apatite crystals subsequently form. However, despite their requisite function in building bone and decades of observations describing intracellular calcium phosphate, the precise role osteoblasts play in mediating bone apatite formation remains largely unknown. To better understand the relationship between intracellular and extracellular mineralization, we combined a sample-preparation method that simultaneously preserved mineral, ions, and ECM with nano-analytical electron microscopy techniques to examine osteoblasts in an in vitro model of bone formation. We identified calcium phosphate both within osteoblast mitochondrial granules and intracellular vesicles that transported material to the ECM. Moreover, we observed calcium-containing vesicles conjoining mitochondria, which also contained calcium, suggesting a storage and transport mechanism. Our observations further highlight the important relationship between intracellular calcium phosphate in osteoblasts and their role in mineralizing the ECM. These observations may have important implications in deciphering both how normal bone forms and in understanding pathological mineralization.     

Benefits in Cognitive Function, Blood Pressure, and Insulin Resistance Through Cocoa Flavanol Consumption in Elderly Subjects With Mild Cognitive Impairment: The Cocoa, Cognition, and Aging (CoCoA) Study

Flavanol consumption is favorably associated with cognitive function. We tested the hypothesis that dietary flavanols might improve cognitive function in subjects with mild cognitive impairment. We conducted a double-blind, parallel arm study in 90 elderly individuals with mild cognitive impairment randomized to consume once daily for 8 weeks a drink containing ≈990 mg (high flavanols), ≈520 mg (intermediate flavanols), or ≈45 mg (low flavanols) of cocoa flavanols per day. Cognitive function was assessed by Mini Mental State Examination, Trail Making Test A and B, and verbal fluency test. At the end of the follow-up period, Mini Mental State Examination was similar in the 3 treatment groups (P=0.13). The time required to complete Trail Making Test A and Trail Making Test B was significantly (P<0.05) lower in subjects assigned to high flavanols (38.10±10.94 and 104.10±28.73 seconds, respectively) and intermediate flavanols (40.20±11.35 and 115.97±28.35 seconds, respectively) in comparison with those assigned to low flavanols (52.60±17.97 and 139.23±43.02 seconds, respectively). Similarly, verbal fluency test score was significantly (P<0.05) better in subjects assigned to high flavanols in comparison with those assigned to low flavanols (27.50±6.75 versus 22.30±8.09 words per 60 seconds). Insulin resistance, blood pressure, and lipid peroxidation also decreased among subjects in the high-flavanol and intermediate-flavanol groups. Changes of insulin resistance explained ≈40% of composite z score variability through the study period (partial r(2)=0.4013; P<0.0001). To the best of our knowledge, this is the first dietary intervention study demonstrating that the regular consumption of cocoa flavanols might be effective in improving cognitive function in elderly subjects with mild cognitive impairment. This effect appears mediated in part by an improvement in insulin sensitivity.     

Dermal innervation in healthy subjects and small fiber neuropathy patients: a stereological reappraisal

The aim of this study was to estimate dermal nerve fiber length (DNFL) using a stereological sampling technique in comparison with a previously reported manual estimation. DNFL was analyzed in skin punch biopsy specimens from 24 healthy volunteers and 18 patients with small fiber neuropathy (SFN) using global spatial sampling that yields unbiased and reliable length estimation. The estimation was carried out in 50‐µm biopsy sections after immunostaining with anti‐protein gene product (PGP) 9.5 antibodies. The length of the PGP9.5‐positive dermal nerves from the dermal–epidermal junction and 200 µm down was measured (DNFL mm^?2). Results were compared with our previously reported manual method. Patients showed a significantly (p < 0.0001) lower DNFL (105 mm^?2 ± 6.4 SD) than healthy subjects (246 mm^?2 ± 8.39 SD). Moderate correlation with age was observed for both healthy subjects (Pearson's r = ?0.33) and patients (Pearson's r = ?0.59). A significant (p < 0.001) correlation between global spatial sampling and manual estimation was observed in both patients and healthy subjects (Pearson's r = 0.62 and 0.61, respectively). These findings provide further evidence on the reliability of dermal nerve morphometry in human skin and strengthen the hypothesis that dermal nerve fibers undergo significant degeneration in SFN.