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

Chuanyi Wang,Yong Wang,Nichole T Huffman,Chaoying Cui,Xiaomei Yao,Sharon Midura,Ronald J Midura,Jeff P Gorski

doi:10.1074/jbc.m805898200

Abstract

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.

Highlights

Schinke et al [4] have proposed that calcification reactions in vivo are passive physiochemical processes occurring readily where local mineralization inhibitors are overwhelmed
We have shown that phosphoglycoprotein BAG-75 delineates future extracellular sites of mineralization in vivo within primary bone and in vitro in osteoblastic cultures termed biomineralization foci [9, 10]
We have previously shown that Maackia amurensis lectin binds to BSP and BAG-75 in Western blots of cell layer extracts of mineralized UMR 106 osteoblastic cells [10]

Summary

Introduction

Schinke et al [4] have proposed that calcification reactions in vivo are passive physiochemical processes occurring readily where local mineralization inhibitors are overwhelmed. We have proposed that mineralization can be divided into a cell-mediated nucleation phase within BMF, followed by passive growth and expansion of these initial crystals (8 –11). In this model, once the initial crystals reach sufficient size and number, the BMF barrier function is abro-. BSP incorporation into BMF peaks at ϳ8 h following phosphate addition, several hours prior to the initial appearance of mineral crystals [18] Based on these findings, we proposed that BMF complexes function in an active mineralization process initiated and controlled by osteoblastic/osteocytic cells.

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