Confocal Laser Raman Microspectroscopy of Biomineralization Foci in UMR 106 Osteoblastic Cultures Reveals Temporally Synchronized Protein Changes Preceding and Accompanying Mineral Crystal Deposition

Abstract

Mineralization in UMR 106-01 osteoblastic cultures occurs within extracellular biomineralization foci (BMF) within 12 h after addition of ␤-glycerol phosphate to cells at 64 h after plating. BMF are identified by their enrichment with an 85-kDa glycoprotein reactive with Maackia amurensis lectin. Laser Raman microspectroscopic scans were made on individual BMF at times preceding (64 -76 h) and following the appearance of mineral crystals (76 -88 h). The range of variation between spectra for different BMF in the same culture was rather small. In contrast, significant differences were observed for spectral bands at 957-960, 1004, and 1660 cm ؊1 when normalized BMF spectra at different times were compared. Protein-dependent spectral bands at 1004 and 1660 cm ؊1 increased and then decreased preceding the detection of hydroxyapatite crystals via the phosphate stretching peak at 959 -960 cm ؊1. When sodium phosphate was substituted for ␤-glycerol phosphate, mineralization occurred 3-6 h earlier. Irrespective of phosphate source, the Raman full peak width at half-maximum ratio for 88 h cultures was similar to that for 10-day-old marrow ablation primary bone. However, if mineralization was blocked with serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 64 -88-h BMF spectra remained largely invariant. In summary, Raman spectral data demonstrate for the first time that formation of hydroxyapatite crystals within individual BMF is a multistep process. Second, changes in protein-derived signals at 1004 and 1660 cm ؊1 reflect events within BMFs that precede or accompany mineral crystal production because they are blocked by mineralization inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. Finally, the low extent of spectral variabilitydetectedamongdifferentBMFatthesametimepointindicatesthat mineralization of individual BMF within a culture is synchronized.

On modeling and nanoanalysis of caries-affected dentin surfaces restored with Zn-containing amalgam and in vitro oral function

“…The1340 cm -1 tracing line exhibited a very low level of noise of fluctuation (Fig 4e). Interestingly, this orientation-sensitive signal decreased when mineralization was allowed to proceed 36 , corroborated by the high Young's modulus values that obtained (Table II (Fig 4c) and 1070 cm -1 (carbonate) (13.89) (Table IVa).…”

“…Monitoring the intensity of this peak has been used to assess the potential increase in phosphate content 36 . Both peaks and areas of phosphate and carbonate groups in CAD also increased in specimens restored with Zn-free amalgams, after 3 w of load cycling.…”

Ions-modified nanoparticles affect functional remineralization and energy dissipation through the resin-dentin interface

“…Similarly, the increases of peaks at 1340 cm -1 (α-helices), after Raman analysis, denoted a greater sensitivity of collagen to molecular orientation, at dentin, in order to enhance further crystallization ). Furthermore, the ratio amide I/amide III raised and Raman CH2 signal diminished at dentin when samples were treated with Ca-NPs after 21 d of SBFS storage, indicating better organization of collagen with improved conformation of the polypeptide chains and lower immature stage of the dentin substrate, respectively (Vanna et al, 2015;Wang et al, 2009;Xu and Wang, 2011) (Table 4). Nevertheless, FWHMC of the carbonate band decreased after 21 d, though MMRC increased (Tables 3a, 3b).…”

Efficacy and micro-characterization of pathophysiological events on caries-affected dentin treated with glass-ionomer cements

“…6. 1340 cm -1 peak: This signal has been assigned to protein α-helices where intensity is sensitive to molecular orientation [35].…”

“…These indexes concerned with the maximum relative degree of mineralization [33,34]. Additionally, peaks at 960 cm and 1450 can be used [35].…”