FOURIER-TRANSFORM INFRARED MICROSPECTROSCOPIC ANALYSIS OF BONES OF OSTEOCALCIN-DEFICIENT MICE PROVIDES INSIGHT INTO THE FUNCTION OF OSTEOCALCIN

Osteocalcin, the gamma-carboxyglutamic acid-containing protein, which in most species is the predominant noncollagenous protein of bone and dentin, has been postulated to play roles in bone formation and remode ling, Recently, genetic studies showed that osteocalcin acts as an inh ibitor of osteoblast function. Based on von Kossa staining and measure ment of mineral apposition rates in tetracycline-labeled bones, osteoc alcin knockout animals were reported to have no detectable alterations in bone mineralization. To test the hypothesis that, in addition to r egulating osteoblastic activity, osteocalcin is involved in regulating mineral properties, a more sensitive assay of mineralization, Fourier transform infrared microspectroscopy (FT-IRM) was used to study thin sections of femora of 4-week-, 6-month- (intact and ovariectomized), a nd 9-month-old wild-type and osteocalcin-knockout mice. FT-IRM spectra provided spatially resolved measures of relative mineral and carbonat e contents, and parameters indicative of apatite crystal size and perf ection, No differences were detected in the mineral properties of the 4-week-old knockout and wild-type mice indicating that the mineralizat ion process was not altered at this time point, Six-month-old wildtype animals had higher mineral contents (mineral:matrix ratios) in cortic al as compared with trabecular bones; mineral contents in knockout and wild-type bones mere not different, At each age studied, carbonate:ph osphate ratios tended to be greater in the wild-type as compared with knockout animals. Detailed analysis of the phosphate nu(1),nu(3) vibra tions in the spectra from 6-month-old wild-type animals indicated that the crystals were larger/more perfect in the cortical as opposed to t he trabecular bones, In contrast, in the knockout animals' bones at 6 months, there were no differences between trabecular and cortical bone in terms of carbonate content or crystallite size and perfection, Spe ctral parameters of the cortical and trabecular bone of the knockout a nimals resembled those in the wild-ape trabecular bone and differed fr om wild-type cortical bone, In ovariectomized 6-month-old animals, the mineral content (mineral:matrix ratio) in the wild-type cortices incr eased from periosteum to endosteum, whereas, in the knockout animals' bones, the mineral:matrix ratio was constant, Ovariectomized knockout cortices had lower carbonate:phosphate ratios than wildtype, and cryst allite size and perfection resembled that in wild-type trabeculae, and did not increase from periosteum to endosteum, These spatially resolv ed data provide evidence that osteocalcin is required to stimulate bon e mineral maturation. (Bone 23:187-196; 1998) (C) 1998 by Elsevier Sci ence Inc. All rights reserved.