Characterization of very young mineral phases of bone by solid state 31Phosphorus magic angle sample spinning nuclear magnetic resonance and X-ray diffraction

Summary The properties of bone mineral change with age and maturation. Several investigators have suggested the presence of an initial or precursor calcium phosphate phase to help explain these differences. We have used solid state ³¹P magic angle sample spinning (MASS) nuclear magnetic resonance (NMR) and X-ray radial distribution function (RDF) analyses to characterize 11-and 17-day-old embryonic chick bone and fractions obtained from them by density fractionation. Density fractionation provides samples of bone containing Ca-P solid-phase deposits even younger and more homogeneous with respect to the age of mineral than the calcium phosphate (Ca-P) deposits in the whole bone samples. The analytical techniques yield no evidence for any distinct phase other than the poorly crystal-line hydroxyapatite phase characteristic of mature bone mineral. In particular, there is no detectable crystalline brushite [DCPD, CaHPO₄ 2H₂O< 1%] or amorphous calcium phosphate (< 8–10%) in the most recently formed bone mineral. A sizeable portion of the phosphate groups exist as HPO₄ ^2– in a brushite (DCPD)-like configuration. These acid phosphate moieties are apparently incorporated into the apatitic lattice. The most likely site for the brushite-like configuration is probably on the surface of the crystals.     

Morphology of bone development and bone remodeling in embryonic chick limbs

Staged embryos from White Leghorn chicken eggs were used to assemble a detailed morphological sequence of events occurring in long bone development from Hamburger-Hamilton stage 32 through stage 44 and 2 days post hatching. The detailed patterning of osteoblasts, osteoid, mineral, and vasculature were observed at the mid-diaphysis of the tibia. At stage 32, the cartilage core is composed of hypertrophie chondrocytes and is surrounded by a continuous ring of mineralized osteoid on which osteoblasts and vasculature reside. At stage 35, the vasculature and associated cell types invade the cartilage core region. By stage 37, marrow occupies the entire cartilage core region at the mid-diaphysis. Anastamosing channels, containing vasculature, interconnect with each other and the marrow region to the inside and the periosteal region to the outside. Clearly, the cartilage is replaced by marrow, not bone.

Mineral deposition at the periosteal surface continues through stage 44 as does mineral resorption on the endosteal surface, although the rate of mineral deposition and resorption varies at different developmental stages. Vasculature plays an important role in the pattern formation of the trabeculae and their channels as can be seen in the developmental sequence within one bone (the tibia) or comparisons between two bones (the tibia and fibula). A model is presented which considers the possibility that osteoprogenitor cells are formed as early as the chondroprogenitor cells. This model also emphasizes the observation that cartilage is not replaced by bone but is replaced by marrow.     

Ultrastructure of matrix vesicles and mineral in unfixed embryonic bone

Femurs of seven-day-old chick embryos were prepared by frozen thin-sectioning and conventional embedding methods and examined by electron microscopy. A collar of bone mineral on the cartilage anlage and a layer of osteoid containing mineral deposits were observed in the frozen thin-sections but not in conventionally prepared tissue. Numerous vesicles and nodules were present in the osteoid and they contained mineral in both the amorphous and crystalline forms. Vesicles and nodules in many stages of mineralization were present and it was possible to arrange them in a sequence of maturation from early, slightly mineralized matrix vesicles to heavily mineralized nodules. This may reflect the sequence of events which occurs in vivo. Crystals within vesicles were frequently found in parallel arrangement. There was no evidence of crystal formation along collagen fibers at this early stage or mineralization. Qualitative electron microprobe analysis of nodules revealed the presence of sodium, magnesium, phosphorus, chlorine, potassium and calcium while adjacent regions of matrix contained the same elements but were deficient in phosphorus.     

Morphological and histochemical events during first bone formation in embryonic chick limbs

Staged embryos from White Leghorn chicken eggs were used to assemble a detailed morphological, cellular and molecular picture of the complex events of first-bone formation. To provide these details, light and electron microscopic, histochemical and immunocytochemical techniques were used to establish a temporal sequence for long bone development in chick wing and leg from Hamburger-Hamilton stage 29 through stage 35. Three distinctive cell regions can be morphologically identified by stage 28 (leg) or 29 (wing) at the middiaphysis. These regions are: 1. an outer grouping of loose mesenchymal and myogenic cells, 2. an osteoprogenitor layer which will later divide to maintain this progenitor layer in a brickwork or stacked configuration and to produce round, tightly packed osteoblasts, and 3. a core (rod) of cartilage. First bone is laid down just outside the cartilage core, initially as a layer of Type I collagen-rich osteoid which later becomes mineralized. Vascular elements then come to reside above this mineral layer, and osteoid is laid down between vascular elements and eventually above them to form a second layer of trabecular bone. As this radial formation of layers of bone is progressing, so too is the proximal and distal expansion of the first bone forming process. A model is presented which considers that chondrogenic and osteogenic cell commitment occur simultaneously in early limb development and that it is the expression of the osteogenic phenotype which governs the boundaries of cartilage development. Importantly, the vasculature plays a key role in the patterning of bone formation well before it enters the cartilaginous core at stage 35 and participates in the erosion of the core. While this report is restricted to events occurring through stage 35, it relies on data presented in a companion report detailing later bone development and remodeling (Pechak et al; Bone 1986) and emphasizes that the cartilage model does not provide the scaffolding for bone but rather defines the marrow space.     

X-ray diffraction studies of the crystallinity of bone mineral in newly synthesized and density fractionated bone

Summary The crystallinity of bone mineral at different stages of maturation has been measured by quantitative X-ray diffraction methods. Crystallinity measurements were made on tibial middiaphyses from 17-day embryonic chicks, newlyformed periosteal bone from embryonic chicks, and density-fractionated bone from post-hatch chickens from 5 weeks to 2 years of age. For a given animal age and degree of mineralization, crystallinity increases with animal age, indicating that changes in bone mineral occur even after mineralization is complete or nearly complete.     

Responsiveness of parathyroid hormone to bone resorption in 13-day-old embryonic chick calvaria cultures

To clarify what kind of process participates in bone resorption, time course of indices of bone resorption was investigated using 13-day-old embryonic chick calvaria. When calvariae were cultured with parathyroid hormone (PTH) at 0.01 U/ml for 8 days, hydroxyproline (Hyp) release was already stimulated by PTH in cultures by 1 to 2 days but stimulation of⁴⁵Ca release was not observed even in cultures by 6 to 8 days. Furthermore, stimulation of collagenase release by PTH was observed prior to that of Hyp release. These results indicate that collagenase release precedes Hyp release, which is followed by⁴⁵Ca release. The release of tartrate-resistant acid phosphatase (TrACP), a marker enzyme of osteoclast, was stimulated by PTH at 0.1 U/ml and above to a greater extent in cultures by 5 to 8 days (phase 11) than in cultures by 0 to 4 days (phase 1). E-64, an inhibitor of cysteine proteinase, inhibited PTH-stimulated⁴⁵Ca release strongly in phase 11 but showed a slight decrease in Hyp release in the same phase. These results suggest that first PTH stimulates collagenase production from osteoblasts, secondly collagenase degrades uncalcified collagen and lastly osteoclasts resorb mineralized bones.     

Growth of embryonic chick tibiae in ovo and in vitro: A scanning electron microscope study

Summary The study describes the ultrastructure of the mineralized portion of chick tibiae from 10 days in ovo to 2 days post-hatch. At 10 days a single mineralized cylinder surrounds the diaphysis. On its outer surface columnar trabeculae join to form ridges parallel to the long axis of the bone. These ridges are covered by another cylinder and form the haversian canals. At 11 days vascular invasion of the marrow cavity occurs and resorption of the endosteal surface begins. This type of periosteal deposition and endosteal resorption is repeated during and subsequent to embryonic development. The mineralized portion of 10-day chick tibiae cultured for 2 days in modified BGJ medium was compared with 10-, 11-, and 12-day tibiae in ovo. Cultured tibiae were similar in length and calcium content to 11-day tibiae in ovo. The form of mineral deposited in ovo and in culture was the same, namely, aggregates of spherical mineral clusters. Differences in culture included the following: (a) few concentric cylinders were deposited as compared with tibiae in ovo; (b) trabeculae were not arranged in rows and ridges in culture; (c) osteocytic lacunae were restricted to bases of trabeculae rather than uniformly distributed as in ovo; and (d) the endosteal surface of tibiae in culture appeared etched.     

An electron probe X-ray microanalytical study of bone mineral in osteogenesis imperfecta

A semiquantitative electron probe X-ray microan-alytical (XRMA) technique, in conjunction with transmission electron microscopy, was used to compare the calcium to phosphorus (Ca/P) molar ratios in calcium phosphate standards of known composition, in normal bone and in bone from patients with osteogenesis imperfecta (OI). Using a modified routine processing and resin embedding schedule, the measured Ca/P molar ratio of calcium phosphates standards of known composition were found to correlate well with the Ca/P molar ratio based on their respective chemical formulae. This technique was then used to compare the Ca/P molar ratio in normal human bone and in OI bone. The Ca/P ratio values for normal bone (Ca/P=1.631) correlated well with those for chemically prepared hydroxyapatite (Ca/P=1.602), but in bone from OI patients, the Ca/P molar ratio was significantly lower (Ca/P=1.488). This study has shown that there is a lower Ca/P molar ratio in OI bone compared with normal, matched bone. This suggests that the mineral deviates from the carbanoapatite usually found in bone. Isomorphous substitutions in the carbanoapatite lattice could account for this although this study has neither proved nor disproved this. The altered bone mineral is an-other factor that could contribute to the increased fracture rate observed in OI.     

The inner surface of chick embryo small intestine by scanning electron microscope

The inner surface of the midgut of chick embryos aged seven to fifteen days of incubation has been examined by scanning electron microscopy. At this period, the surface of the mucous membrane undergoes significant morphological changes; in fact, while at seven days of incubation it appears to be smooth and regular, in the following days it begins to show longitudinal folds increasingly higher, more numerous and complicated. After eleven days of incubation some folds take a zig-zag appearance that progressively becomes more evident and extends to all the folds. In the mean time, the enterocytes undergo a gradual differentiation and represent the only type of epithelial cells at this stage of development. Their apex until seven days of incubation is dome-shaped, provided with short microvilli and separated from the surrounding cells by deep circular grooves. At about thirteen days the apex appears to be less swollen, with longer and more numerous microvilli and delimited by microplicae arranged in a hexagonal disposition.     

Gene polymorphisms,bone mineral density and bone mineral content in young children:the Iowa bone development study

We examined the association of candidate gene polymorphisms with bone mineral density (BMD) and bone mineral content (BMC) in a cohort of 428 healthy non-Hispanic white children participating in the Iowa Bone Development Study, a longitudinal study of determinants of bone accrual in childhood. BMD and BMC measurements of the hip, spine and whole body were made using a Hologic 2000 Plus densitometer in 228 girls and 200 boys ages 4.5–6.5 years. Genotypes at 14 loci representing eight candidate genes [type I collagen genes (COL1A1 and COL1A2), osteocalcin, osteonectin, osteopontin, vitamin D receptor (VDR), estrogen receptor (ER), androgen receptor (AR)] were determined. Gender-specific and gender-combined prediction models for bone measures that included age, weight, height (and gender) were developed using multiple linear regression analysis. COL1A2 and osteocalcin genotypes were identified as having the strongest and most consistent association with BMD/BMC measures. Osteonectin, osteopontin and VDR translation initiation site polymorphisms were associated with some individual bone measures, but none of the associations was as consistent as those identified for the COL1A2 and osteocalcin genes. No association was identified with COL1A1 (RsaI and Sp1), VDR (BsmI) and ER polymorphisms (PvuII, XbaI, TA) and BMD/BMC. However, we identified significant gene-by-gene interaction effects involving the ER and both VDR and osteocalcin, which were associated with BMD/BMC. Our data suggest that genetic variation at multiple genetic loci is important in bone accrual in children. Moreover, the combination of genotypes as several loci may be as important as a single genotype for determining BMD and BMC.     

The effect of grinding of human dentine examined by X-ray diffraction,infrared spectroscopy,and electron microscopy

Analysis of human dentine by infrared spectrophotometry suggests that ball-grinding may result in damage of the apatite crystallites. The present study includes further assessments of this effect by X-ray diffraction, infrared spectroscopy, and electron microscopy. Each of three coarse-ground dentine samples (Group I) was divided into three portions of 30 mg. One of these portions was ball-ground for approximately 1 min (Group II), the second portion for 6 min (Group III), and the third portion for 23 min (Group IV). The 002 reflection showed line broadening, most marked from Group II to III. Electron microscopy showed a gradual change in crystallite appearance with increased grinding, most pronounced from Group II to III. These observations indicate that by prolonged grinding a limit is approached where no further changes in the crystallites occur. Electron microscopy also indicated that fracture of the crystallites might have occurred. This was probably accompanied by strains in the lattice. The infrared spectra indicated that no breakdown of the apatite structure had occurred during the entire grinding.     

辣木发酵物对大鼠生长、骨骼代谢和骨密度的影响

目的研究辣木发酵物对大鼠生长发育、骨骼代谢和骨密度的影响。方法选用24只体重(74.38±0.63)g、生长状况良好的清洁级雄性SD大鼠,随机分为实验组和对照组,每组3个重复,每个重复4只。对照组饲喂基础日粮,实验组在基础日粮中添加0.5%辣木发酵物,试验期10周。测量大鼠体重、采食量、股骨湿重、干重和股骨长,血清中钙、磷含量和碱性磷酸酶活性以及腰椎骨骨密度(BMD)。结果试验组和对照组采食量无明显差异(P0.05),实验组大鼠体重较对照组有显著增加(P0.01);与对照组相比,实验组大鼠的股骨湿重显著增加(P0.05),股骨长与腰椎骨密度有极其显著的提高(P0.01),股骨干重有增加的趋势(P=0.0538);实验组血清钙含量显著低于对照组(P0.01),血清磷含量显著高于对照组(P0.05),两组碱性磷酸酶活性无显著差异(P0.05)。结论日粮中添加辣木发酵物能够促进大鼠生长发育,促进大鼠骨骼钙的沉积和骨骼生长,增强骨骼强度。     

The effect of grinding of human dentine examined by X-ray diffraction, infrared spectroscopy, and electron microscopy

Analysis of human dentine by infrared spectrophotometry suggests that ball-grinding may result in damage of the apatite crystallites. The present study includes further assessments of this effect by X-ray diffraction, infrared spectroscopy, and electron microscopy. Each of three coarse-ground dentine samples (Group I) was divided into three portions of 30 mg. One of these portions was ball-ground for approximately 1 min (Group II), the second portion for 6 min (Group III), and the third portion for 23 min (Group IV). The 002 reflection showed line broadening, most marked from Group II to III. Electron microscopy showed a gradual change in crystallite appearance with increased grinding, most pronounced from Group II to III. These observations indicate that by prolonged grinding a limit is approached where no further changes in the crystallites occur. Electron microscopy also indicated that fracture of the crystallites might have occurred. This was probably accompanied by strains in the lattice. The infrared spectra indicated that no breakdown of the apatite structure had occurred during the entire grinding.     

Light and electron microscopic demonstration of osteocalcin antigenicity in embryonic and adult rat bone

Of the noncollagenous proteins in bone, about 20% consists of osteocalcin. This vitamin K-dependent protein can be found in adult bone, but its presence in embryonic bone could not be demonstrated unequivocally by biochemical methods. Therefore, we used light and electron microscopic immunohistochemical methods to investigate whether osteocalcin antigenicity could be demonstrated in radii of 20-day-old rat embryos. The results show that osteocalcin antigenicity can be demonstrated in the bone matrix of adult bone and in the shaft and endochondral bone matrix of embryonic bone. It could not be demonstrated in calcified cartilage matrix. In bone the antigenicity was observed in the early foci of calcification, i.e., the mineralization nodules.     

去卵巢对大鼠皮质骨微结构、骨密度及生物力学的影响

目的 应用显微CT和四点弯曲实验观察去卵巢对大鼠股骨皮质骨骨密度、微结构及生物力学性能的影响.方法 24只7月龄雌性SD大鼠随机分为去卵巢(OVX)组和假手术(Sham)组,于手术后3周、15周各处死6只,显微CT扫描左侧股骨中段.右侧股骨行四点弯曲试验.结果 去卵巢3周至15周,OVX组股骨皮质骨内径周长、外径周长、皮质骨面积、骨髓腔面积、截面总面积骨密度、最大力、弹性模量、断裂强度与Sham组相比无明显差异(P＞0.05).OVX组15周弹性模量比3周低36％ (P ＜0.05).结论 去卵巢15周内大鼠股骨中段皮质骨微结构及其骨密度和股骨力学性能无明显变化,但去卵巢组股骨生物力学性能减退较快.