Confocal laser Raman microspectroscopy of biomineralization foci in UMR 106 osteoblastic cultures reveals temporally synchronized protein changes preceding and accompanying mineral crystal deposition.
Mineralization in UMR 106-01 osteoblastic cultures occurs within extracellular biomineralization foci (BMF) within 12 h after addition of beta-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 beta-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 variability detected among different BMF at the same time point indicates that mineralization of individual BMF within a culture is synchronized.
Wang
Chuanyi
C
Biomaterials Section, Department of Oral Biology, School of Dentistry, University of Missouri, Kansas City, Missouri 64108, USA.
Wang
Yong
Y
Huffman
Nichole T
NT
Cui
Chaoying
C
Yao
Xiaomei
X
Midura
Sharon
S
Midura
Ronald J
RJ
Gorski
Jeff P
JP
D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D017886:Durapatite / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010742:Phospholipid Ethers / Q000494:pharmacology; D010835:Phytohemagglutinins / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013059:Spectrum Analysis, Raman / Q000379:methods; D013997:Time Factors
D010742:Phospholipid Ethers; D010835:Phytohemagglutinins; D015842:Serine Proteinase Inhibitors; C100156:leukoagglutinins, plants; D017886:Durapatite