Metastable Equilibrium Solubility Behavior of Carbonated Apatite in the Presence of Solution Strontium

The purpose of this study was to use the concept of metastable equilibrium solubility (MES) to describe the anomalous solubility behavior of carbonated apatite (CAP) in the presence of solution strontium. A CAP sample (4.8 wt% CO(3), synthesized at 70 degrees C) was prepared by precipitation. Baseline MES distributions were determined in a series of 0.1 M acetate buffers containing only calcium and phosphate (no strontium) over a broad range of solution conditions. In order to assess the influence of strontium, MES profiles were then determined in a similar fashion with 20, 30, 40, 50, 60, 70, and 80% of the solution calcium being replaced on an equal molar basis by solution strontium. From the compositions of the equilibrating buffer solutions, ion activity products (IAPs) of the form Ca(10-n)Sr(n)(PO(4))(6)(OH)(2) (n = 0-10) were calculated in an attempt to determine the correct function governing the dissolution of the CAP preparation. The results demonstrate the following important findings: (a) at high solution strontium/calcium ratios (i.e., when 60% or more of the solution calcium was replaced by strontium), the MES profiles in all the experiments were found to be essentially superimposable when the solution IAPs were calculated using the stoichiometry of Ca(6)Sr(4)(PO(4))(6)(OH)(2), and (b), at low solution strontium/calcium ratios (i.e., when 40% or less of the solution calcium was replaced by strontium), the stoichiometry yielding MES data superpositioning was found to be that of hydroxyapatite. When other stoichiometries were assumed, good superpositioning of the data was not possible.     

Metastable Equilibrium Solubility Behavior of Carbonated Apatite in the Presence of Solution Strontium

The purpose of this study was to use the concept of metastable equilibrium solubility (MES) to describe the anomalous solubility behavior of carbonated apatite (CAP) in the presence of solution strontium. A CAP sample (4.8 wt% CO₃, synthesized at 70°C) was prepared by precipitation. Baseline MES distributions were determined in a series of 0.1 M acetate buffers containing only calcium and phosphate (no strontium) over a broad range of solution conditions. In order to assess the influence of strontium, MES profiles were then determined in a similar fashion with 20, 30, 40, 50, 60, 70, and 80% of the solution calcium being replaced on an equal molar basis by solution strontium. From the compositions of the equilibrating buffer solutions, ion activity products (IAPs) of the form Ca_10-nSr_n(PO₄)₆(OH)₂ (n = 0–10) were calculated in an attempt to determine the correct function governing the dissolution of the CAP preparation. The results demonstrate the following important findings: (a) at high solution strontium/calcium ratios (i.e., when 60% or more of the solution calcium was replaced by strontium), the MES profiles in all the experiments were found to be essentially superimposable when the solution IAPs were calculated using the stoichiometry of Ca₆Sr₄(PO₄)₆(OH)₂, and (b), at low solution strontium/calcium ratios (i.e., when 40% or less of the solution calcium was replaced by strontium), the stoichiometry yielding MES data superpositioning was found to be that of hydroxyapatite. When other stoichiometries were assumed, good superpositioning of the data was not possible.     

Relationships Involving Metastable Equilibrium Solubility, Surface Complexes, and Crystallite Disorder with Carbonated Apatites

Previous studies have shown that the metastable equilibrium solubility (MES) behavior of carbonated apatites (CAPs) may be described by a surface complex with the hydroxyapatite (HAP) stoichiometry in the absence of solution fluoride and by that with the fluorapatite (FAP) stoichiometry when appreciable solution fluoride is present. Studies have also shown that the magnitude of the MES is directly related to the crystallinity of the CAP. The aim of the present investigation was to examine the relationship between the CAP MES determined in the presence of solution fluoride and CAP crystallinity and to examine the effect of the change in the stoichiometry of the surface complex (from that of HAP to that of FAP) upon the relationship of the CAP MES to crystallinity. CAP samples were prepared by methods based on the precipitation of CAP from calcium phosphate solutions and the hydrolysis of dicalcium phosphate dihydrate in bicarbonate solutions. From X-ray diffraction experiments, the crystallite microstrain, and the full width at half maximum (FWHM) of the 002 reflection were determined for the CAPs. From CAP MES experiments conducted in the presence of solu tion fluoride, linear plots of the mean MES (i.e., mean pK(FAP)) values vs the crystallinity parameter (i.e., microstrain and FWHM) were obtained that yielded slopes that were essentially the same as those obtained in the absence of solution fluoride (i.e., mean pK(HAP) values vs crystallinity). This parallel finding suggests that the CAP crystallite disorder affects the energetics of the two surface complexes essentially to the same extent and provides new insight into the nature of CAP surface complexes.     

Relationships Among Carbonated Apatite Solubility, Crystallite Size, and Microstrain Parameters

The use of the metastable equilibrium solubility (MES) concept to describe the solubility properties of carbonated apatites (CAPs) and human dental enamel (HE) has been well established in previous studies using a range of CAPs with varying carbonate contents and crystallinities. It was shown in these studies that the mean value of the CAP MES is directly related to the broadening parameter full width at half maximum (FWHM) of the 002 reflection of the X-ray diffraction profile. The apparent solubility of the CAPs increased monotonically with an increase in the broadening of the diffraction peaks, and when this peak broadening was taken into account, carbonate had no additional effect upon the MES. The broadening of the diffraction peaks has been used as an indicator of crystallinity, and is generally influenced by both crystallite size and microstrain. The purpose of the present study was to extract the crystallite size and microstrain parameters separately from the X-ray diffraction peaks and then to determine their relationships to the corresponding MES values. The samples studied were CAPs synthesized by precipitation from Ca(NO₃)₂ and NaH₂PO₄ solutions in carbonate containing media at temperatures of 95, 80, and 70°C, and powdered HE. The crystallite size and microstrain parameters were determined simultaneously with the refinement of the structural parameters with the Rietveld method of whole-pattern-fitting structure-refinement. A modified pseudo-Voigt function was used to model the observed peak profiles. The MES distributions for the CAPs and HE were determined by a previously described method. The results of this study showed that the CAPs possessed an MES distribution and therefore provided further support that MES distribution is a common phenomenon, regardless of the method of CAP synthesis. The crystallite size decreased and the microstrain increased with increasing carbonate content and decreasing temperature of synthesis of the CAPs. A plot of the mean of the MES distribution versus the microstrain parameter showed that the apparent solubility of the CAPs and HE correlated very well with the microstrain parameter. On the other hand, a plot of the mean of the MES distribution versus the crystallite size parameter showed a poor correlation between MES and crystallite size. These findings support a view that microstrain, rather than crystallite size, is the dominant factor governing the effective solubility of the CAPs and dental enamel. Received: 24 March 1998 / Accepted: 1 October 1998     

MicroRaman Spectral Study of the PO4 and CO3 Vibrational Modes in Synthetic and Biological Apatites

The carbonate and phosphate vibrational modes of different synthetic and biological carbonated apatites were investigated by Raman microspectroscopy, and compared with those of hydroxyapatite. The ν₁ phosphate band at 960 cm⁻¹ shifts slightly due to carbonate substitution in both A and B sites. The spectrum of type A carbonated apatite exhibits two ν₁ PO₄ ³⁻ bands at 947 and 957 cm⁻¹. No significant change was observed in the ν₂ and ν₄ phosphate mode regions in any carbonated samples. The ν₃ PO₄ ³⁻ region seems to be more affected by carbonation: two main bands were observed, as in the hydroxyapatite spectrum, but at lower wave numbers. The phosphate spectra of all biominerals apatite were consistent with type AB carbonated apatite. In the enamel spectrum, bands were observed at 3513 and at 3573 cm⁻¹ presumably due to two different hydroxyl environments. Two different bands due to the carbonate ν₁ mode were identified depending on the carbonate substitution site A or B, at 1107 and 1070 cm⁻¹, respectively. Our results, compared with the infrared data already reported, suggest that even low levels of carbonate substitution induce modifications of the hydroxyapatite spectrum. Increasing substitution ratios, however, do not bring about any further alteration. The spectra of dentine and bone showed a strong similarity at a micrometric level. This study demonstrates the existence of acidic phosphate, observable by Raman microspectrometry, in mature biominerals. The HPO₄ ²⁻ and CO₃ ²⁻ contents increase from enamel to dentine and bone, however, these two phenomena do not seem to be correlated. Received: 5 January 1998 / Accepted: 12 May 1998     

EPR Properties of Synthetic Apatites, Deorganified Dentine, and Enamel

Electron paramagnetic resonance spectroscopy (EPR) was used to study synthetic hydroxyapatite and ∼1, 2, and 6% synthetic carbonated apatites, deorganified dentine, and enamel. The carbonated apatites were synthesized by hydrolysis of dicalcium phosphate. Comparisons were made with spectra from enamel and deorganified dentine. Microwave power saturation and dose responses were determined for the synthetic materials. The Marquardt version of the Levenberg decomposition method was used to extract individual signals from the apatite data. Two samples of dentine were irradiated with 25 and 100 Gy, respectively, from a ⁶⁰Co source. The first sample was then deorganified at 200°C using the Soxhlet extraction technique. A third sample was irradiated with 100 Gy after deorganification. The resulting EPR spectra were then compared. It was determined that the dosimetric signal of 2% synthetic carbonated apatite was approximately the same as that of enamel. It was also verified that the dosimetric signal saturates at about 2% in synthetic carbonated apatites. The study established that the precenters responsible for the dosimetric signal (g_⊥= 2.0018, g_∥= 1.9985) are preferentially concentrated in the surface-accessible region of the mineral component, as shown by the approximately 80% attenuation of the dosimetric signal in dentine following deorganification. The precenters responsible are not destroyed by the deorganification since the magnitude of the dosimetric signal from the dentine specimen irradiated following deorganification was approximately twice that of the comparable untreated, irradiated sample. Finally, the dose response of 2 and 6% synthetic carbonated apatites was determined. Received: 2 May 1996 / Accepted: 26 June 1997     

Effect of Age on Rat Bone Solubility and Crystallinity

It has been shown that biominerals such as dental enamel and bone demonstrate a non-thermodynamic equilibrium state following initial dissolution where no further mineral dissolution or precipitation occurs; this state is termed metastable equilibrium solubility (MES). Furthermore, these minerals are composed of a distribution of domains each with their own MES. Recent studies have also demonstrated a linear relationship between crystallinity and the mean MES of these minerals, with solubility decreasing with increasing crystallinity. This study investigates the effect of age on the MES and crystallinity of rat bone mineral. The bone mineral samples were prepared by protein extraction with a series of hydrazine and alcohol solutions. The MES distributions of the bone mineral were measured by exposure to 0.1 M acetate buffers containing a range of calculated amounts of calcium (Ca) and phosphate for 48 h. The amount of mineral dissolved in each solution was determined from Ca and phosphate analyses of the undissolved residue. The full width of the half maximum (FWHM) of the 002 reflection of the X-ray powder diffraction was used as an indicator of crystallinity. The MES of mineral from bone of rats of different ages (1-25 months) were compared. Results of this study indicate that (l) there is a difference in the mean MES of bone mineral from rats of different ages, with older bone mineral being less soluble and more crystalline than bone mineral from younger rats; (2) the nature of the solubility distribution changes from a narrow to a broader distribution with age; and (3) all of the bone samples demonstrated an inverse-linear correlation between crystallinity and mean MES value consistent with previous results obtained with synthetic apatites and dental enamel.     

Effects of Bone CS-Proteoglycans, DS-Decorin, and DS-Biglycan on Hydroxyapatite Formation in a Gelatin Gel

The small leucine-rich bone proteoglycans, biglycan and decorin, can be purified by chromatography on hydroxyapatite columns, demonstrating their potential affinities for bone apatite. To determine their effects on in vitro apatite formation and growth, a mixture of the chondroitin-sulfate (CS) bone proteoglycans, or purified fractions of the dermatan sulfate (DS) containing proteoglycans, DS-decorin and DS-biglycan obtained from skin and articular cartilage, respectively, were analyzed in a gelatin gel diffusion system in which apatite formation occurs in the absence of proteins in a 3.5 day period. Low concentrations of the bone CS-proteoglycan mixture and low DS-biglycan concentrations (5–25 μg/ml) increased apatite formation relative to proteoglycan-free controls at 3.5 days. The CS-proteoglycan mixture was less effective at 50 μg/ml than at 10 μg/ml. DS-biglycan was similarly most effective at 5–25 μg/ml. At 5 days, when apatite growth and proliferation were assessed, 10 and 50 μg/ml of both CS-bone proteoglycan and DS-biglycan increased mineral yields. DS-decorin, in contrast, had no significant effect on mineral accumulation at any of these concentrations. In seeded growth experiments, 1 and 10 μg/ml CS-proteoglycan and 10 and 50 μg/ml DS-biglycan were significant effective inhibitors of mineral accretion, whereas DS-decorin showed no tendency to inhibit seeded growth. Using molar extinction coefficients to determine concentrations, the binding of DS-biglycan and DS-decorin to apatite (specific surface 54 m²/g) was determined using a Langmuir adsorption isotherm model. DS-biglycan had a greater affinity for apatite than DS-decorin (0.285 ml/μmol versus 0.0098 ml/μmol). DS-biglycan binding was more specific with fewer binding sites (3.5 μmol/m² compared with 18.2 μmol/m² for DS-decorin). Data suggest that of the small proteoglycans, biglycan may play a more significant role than decorin in the regulation of mineralization. Received: 10 June 1996 / Accepted: 25 April 1997     

Effect of fluoride pretreatment on the solubility of synthetic carbonated apatite

The purpose of this research was to address the following question: How is the solubility of fluoride-pretreated carbonated apatite (CAP) in aqueous acidic media related to the equilibrium solution fluoride and/or the CAP adsorbed fluoride levels? A CAP sample prepared by a precipitation method at 70 degrees C containing approximately 6% carbonate was fluoride-treated (F adsorption from neutral aqueous solutions) to yield a approximately 1000 ppm F CAP and a approximately 3300 ppm F CAP. Metastable equilibrium solubility distributions were determined in acetate buffers at pH 5.0. Solution fluoride, calcium, phosphate, and pH were determined from the equilibrated solutions. The equilibrium solution fluoride levels were extremely low, e.g., as low as approximately 0.10 ppb to approximately 0.30 ppb at 50% dissolved for the two CAP preparations. The approximately 3300 ppm F CAP yielded a lower solubility than the approximately 1000 ppm F CAP (shift in the mean pKHAP value of 1.5-2 units). This can be attributed to the lower solution F(-) for the sample containing approximately 1000 ppm fluoride compared with the approximately 3300 ppm fluoride-containing CAP. These important findings suggest that a fluoride treatment simply may provide an adsorption fluoride depot for subsequent release, providing a solution fluoride effect upon the CAP solubility and not necessarily any intrinsic alteration of the mineral solubility.     

Matrix Vesicles Promote Mineralization in a Gelatin Gel

Extracellular matrix vesicles (MVs) are associated with initial calcification in a variety of tissues, but the mechanisms by which they promote mineralization are not certain. In this study, MVs isolated from fourth passage rat growth plate chondrocyte cultures were included within a gelatin gel into which calcium and phosphate ions diffused from opposite ends. In this gel, apatite formation occurs by 3.5 days in the absence of mineralization promoters, allowing measurement of the ability of different factors to ``nucleate' apatite before this time or to assess the effects of molecules which modulate the rate and extent of mineral deposition. Mineral ion accumulation and crystal type are assayed at 5 days. In this study, MV protein content in the central band of a 10% gelatin gel was varied by including 100 μl of a Tris-buffered solution containing 0–300 μg/ml MV protein. There was a concentration-dependent increase in mineral accretion. Whereas 10 μg MV protein in the gel did not significantly promote apatite formation as compared with vesicle-free gels, 20 and 30 μg MV protein in the gel did promote apatite deposition. Inclusion of 10 mM β-glycerophosphate in the gels, along with MVs, did not significantly increase apatite formation despite the demonstrable alkaline phosphatase activity of the MVs. In contrast, MVs at all concentrations significantly increased apatite accumulation when proteoglycan aggregates or ATP, inhibitors of apatite formation and proliferation, were included in the gel. Slight increases in calcium, but not phosphate accumulation, were also noted when an ionophore was included with the MVs to facilitate Ca ion transport into the vesicles. FT-IR analysis of the mineral formed in the vesicle-containing gels revealed the presence of a bone-like apatite. These data suggest that MVs facilitate mineralization by providing enzymes that modify inhibitory factors in the extracellular matrix, as well as by providing a protected environment in which mineral ions can accumulate. Received: 28 January 1996 / Accepted: 9 August 1996     

Acellular Mineral Deposition in Collagen-Based Biomaterials Incubated in Cell Culture Media

Rapid developments in tissue engineering have renewed interest in biodegradable three-dimensional structures such as collagen-based biomaterials. Collagen matrices seeded in vitro with fibroblasts, osteoblasts, and chondrocytes can form tissues resembling skin, bone, and cartilage that could be used as functional substitutes for damaged tissues. Collagen is associated with both dystrophic calcification of collagenous implants and bone mineralization. We report here the calcification properties of collagen sponges incubated in cell-free media. Mineral deposited in sponges was identified by X-ray and electron diffraction, Fourier transform infrared spectroscopy, and the molar ratio of calcium:phosphorus (Ca:P) as a poorly crystalline apatite similar to bone. The degree of calcification increased with length of incubation and the Ca and P content of the media, with 10–15% Ca (dry weight) after 21 days' incubation in media containing 1.6–3 mM Ca and a Ca × P molar product of 2–3 mM², but only 2% Ca after incubation in medium with 1.33 mM Ca and a 1.7 mM² Ca × P molar product. Mineral deposition was completely inhibited in sponges that were washed extensively and initially contained less than 0.01% P. Readdition of phosphate in these sponges and subsequent freeze drying and sterilization restore their mineralization capacity, suggesting that collagen per se cannot initiate calcification and that the inorganic phosphate content associated with the collagen preparation process is in the solid state a potential nucleator. Addition of chondroitin 4-sulfate to the sponges partially or totally inhibited mineral deposition, even though 80–90% of the compound was released within 24 hours. These results indicate that acellular calcification of collagen-based biomaterials can occur under the culture conditions currently used in tissue engineering. Received: 30 October 1997 / Accepted: 30 September 1999     

Stable Transfection of Nonosteogenic Cell Lines with Tissue Nonspecific Alkaline Phosphatase Enhances Mineral Deposition Both in the Presence and Absence of β-Glycerophosphate: Possible Role for Alkaline Phosphatase in Pathological Mineralization

It is documented that alkaline phosphatase (AP) plays an important role in bone mineralization. Considering that TN-AP is expressed in periodontal ligament fibroblasts, renal epithelial cells, and vascular endothelial cells, and that TN-AP is both a calcium-/phosphate-binding protein and a phosphohydrolytic enzyme, we hypothesize that membrane-bound AP also plays an important role in the initiation of physiological and pathological mineralizations in tissues other than bone and cartilage. To test this hypothesis, nonosteoblast cell lines, including a fibroblast line, a renal epithelial line, and a capillary endothelial line, were stably transfected to express high levels of rat bone AP on their cell surfaces. These rat bone AP-expressing cells were then cultured on filter membranes in the presence or absence of β-glycerol phosphate. von Kossa staining for calcium phosphate and transmission electron microscopy with electron diffraction analysis for minerals were employed to investigate the effect of membrane AP on extracellular calcium phosphate mineralization. Our results indicated that AP expression on these nonosteoblast-like cell surfaces have induced extracellular hydroxyapatite (HAP) mineralization. Our findings support the concept that membrane-bound AP contributes to extracellular apatitic mineralization by mechanisms that do not necessarily involve its hydrolase activity. They also suggest that AP might be important for the initiation of pathological mineralization in nonosteogenic tissues. Received: 11 January 1996 / Accepted: 31 October 1996     

Ultrastructural Study of Apatite Precipitation in Implanted Calcium Phosphate Ceramic: Influence of the Implantation Site

Macroporous biphasic calcium phosphate (MBCP) blocks were implanted into rabbit trabecular bone and muscle, recovered 18 weeks later, and then observed and analyzed by transmission electron microscopy (TEM), electron diffraction, and electron microprobes. The results showed that (1) apatitic microcrystals appeared by secondary nucleation in both bone and muscle sites; (2) precipitated microcrystals were aggregated around ceramic crystals in bone sites but distributed randomly and without orientation in micropores in muscle sites; (3) the ratio of calcium to phosphorus was higher for microcrystals in bone than muscle sites; and (4) precipitated microcrystals around β-tricalcium phosphate (β-TCP) crystals were less aggregated and dense than those around hydroxyapatite (HA). These findings suggest that microenvironmental parameters such as fluid circulation and the interaction of ceramics with proteins or cells affect the physicochemical dissolution/reprecipitation process. Epitaxic growth of apatitic microcrystals seems more favorable from HA than β-TCP. Received: 18 October 1996 / Accepted: 20 November 1997     

The Effect of Fluoride Treatment on Bone Mineral in Rabbits

Fluoride therapy has been used clinically for many years, but its use remains controversial and many basic questions remain unanswered. Accordingly, this study returns to an animal model to study the effects of high doses of fluoride on bone mineral in rabbits. Twelve rabbits, aged 3(1/2) months at the start of the study, received drinking water fluoridated at 100 ppm while their 12 control counterparts drank distilled water. All rabbits were sacrificed after 6 months. Fluoride was readily incorporated into femoral cortical bone (7473 +/- 966 ppm F versus 1228 +/- 57 ppm in controls; P < 0.00005). Fluoride therapy led to increased mineralization, as measured by density fractionation (P < 0.0005 for the distributions). The bone mineral itself was altered, with a significant increase in the width of crystals (66.2 +/- 2.0 A versus 61.2 +/- 0.9 A; P < 0.01). The microhardness of both cortical and cancellous bone in the femoral head of fluoride-treated rabbits was greater than that in the controls (P < 0.05). The phosphate, calcium, and carbonate contents in the bone was the same in both groups. Finally, fluoride administration did not affect the architecture or connectivity of cancellous bone in the femoral head. Previously published data [1] indicated that the mechanical properties of bone were adversely affected; this suggests that the effect of high doses of fluoride on the strength and stiffness of bone may be mediated by its effect on bone mineral.     

Ultrastructural and Electron Diffraction of the Bone-Ceramic Interfacial Zone in Coral and Biphasic CaP Implants

We investigated the influence of natural coral implants used as a bone substitute on the quality of bone ingrowth in rabbits 2, 3, and 6 weeks after implantation. Explants were characterized by transmission electron microscopy and electron diffraction. Bone ingrowth has been previously demonstrated by light microscopy, however, few have been performed in electron microscopy to compare mineralized tissue ingrowth in coral implants which occurs at the expense of calcium carbonate to that of calcium phosphate (CaP) implants. The interface between coral aragonite and mineralized tissue or bone was abrupt, with no invasion of the aragonite structure by newly formed crystals, as occurs in micropores when biphasic CaP (BCP) ceramics were used. The restoring process appears to be different from that induced by BCP implants. Precipitation of needle-like apatite crystals on the CaCO₃ implant surface was not observed. Instead, apatitic smooth-shaped crystals formed in aggregates. The coral dissolution process does not release phosphate and so precipitation of apatite does not occur in the micropores of the coral implant, thereby limiting the formation of an apatite layer and hence bone bonding to the outer surface of the implant. In addition, on the outer surface of the implant, close to bone and a phosphorus source, the CaP crystals that do form are in aggregates presumably due to the carbonate and mismatch between the aragonite and the apatite. This seems to result in a delayed bone attachment or weaker bone bonding than CaP implants which encourage an epitaxial biological crystal deposition. Received: 21 July 1995 / Accepted: 7 August 1997     

Matrix Vesicle Calcification in Bones of Adult Rats

To clarify the calcification mechanism that functions in bone formation in adult rats, the ultrastructure of tibial trabeculae and calvarial endostea obtained from 8- to 18-month-old rats was investigated morphologically, and compared with that of 19.5-day post-coitum fetal rats. In both samples, osteoid was observed between the activated osteoblasts and the calcified matrix, which contained matrix vesicles enclosed by a biological membrane. Some of these vesicles contained needle-like crystals thought to be hydroxyapatite, suggesting probable matrix vesicle calcification. These results indicate that matrix vesicle function not only in the initial calcification that occurs during embryonic ossification but also contribute to bone formation in adults. Received: 16 September 1998 / Accepted: 3 January 2000     

Apatite Mineralization in Teeth of the Chiton Acanthopleura echinata

Raman spectroscopy has been used to demonstrate, for the first time, that calcium mineralization in the core of the major lateral teeth of the chiton Acanthopleura echinata takes place as an ordered process, with crystalline carbonated apatite being the first mineral deposited. Deposition begins at the top of the tooth core, under the so-called tab region, progresses down the interior surface of the tab and lepidocrocite layer, and then extends outwards to the anterior surface. Mineralization is not initiated until the lepidocrocite layer has isolated the core of the tooth from the magnetite cap. The last region to be infiltrated is the anterior basal region of the tooth cusp, immediately above the junction zone. The junction zone is also a region of high ion density, as determined by energy dispersive spectroscopy (EDS) analysis, but we show here for the first time that it is free of mineral deposits, acting instead as a transfer and storage region. Received: 7 January 2000 / Accepted: 30 May 2000 / Online publication: 2 November 2000     

Anionic Effects on the Size and Shape of Apatite Crystals Grown from Physiological Solutions

Comparatively little is known of the role tissue fluid electrolytes have in establishing the size and shape of apatite crystals deposited in skeletal tissues. In vitro accretion experiments using synthetic apatite seed crystals comparable in size to bone apatite were performed to assess the extent to which these crystalline features may be affected by direct electrolyte/mineral interactions. A constant composition method was used to maintain the accretion reactions under physiological-like solution conditions (1.33 mmol/liter Ca²⁺, 1.0 mmol/liter total inorganic phosphate, (0 or 26) mmol/liter carbonate, 270 mmol/kg osmolality, pH 7.4, 37°C). When the mass of the new accretions equaled the initial seed mass, the solids were harvested and the net change in crystal size resulting from the new accretions was assessed by X-ray diffraction line-broadening analysis. All the electrolytes examined in this study inhibited the accretion rate. The order of effectiveness was phosvitin > polyaspartate ≈ polyglutamate > 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) > bovine serum albumin (BSA) > citrate. Citrate and BSA also reduced the mean crystal size of the harvested solids compared with those harvested in the absence of added electrolyte, a finding that suggests that citrate and BSA suppressed growth of the seed crystals in favor of the proliferation of new smaller crystals. In contrast, a net increase in size following accretion compared with controls suggests that the other more strongly inhibiting electrolytes stimulated growth of the primary seed crystals and/or of the secondary crystals. These size changes, however, were anisotropic, with the anions effecting primarily increases in crystal width/thickness rather than in length. The effects were also more pronounced in the presence of carbonate. Our findings suggest that the strength of the interaction with the crystal surface may be relatively more important than molecular size or conformational complexity in establishing the effect that electrolytes have on apatite growth and proliferation. The results also suggest that adsorbed electrolytes may be a significant factor in controlling the size of apatite crystals in skeletal tissues by inducing proliferation of new crystals and/or affecting crystal shape by selectively modifying growth of the lateral dimensions. Received: 28 June 1999 / Accepted: 3 January 2000     

Bone Response to In Vivo Mechanical Loading in C3H/HeJ Mice

Bone, being sensitive to mechanical stimulus, adapts to mechanical loads in response to bending or deformation. Although the signal/receptor mechanism for bone adaptation to deformation is still under investigation, the mechanical signal is related to the amount of bone deformation or strain. Adaptation to changes in physical activity depends on both the magnitude of increase in strain above average daily levels for maintaining current bone density and the Minimum Effective Strain (MES) for initiating adaptive bone formation. Given the variation of peak bone density that exists in any human population, it is likely that variation in levels for MES is, to a considerable degree, inherited and varies among animal species and breeds. This study showed a dose-related periosteal response to loading in C3H/HeJ mice. The extent of active formation surface, the rate of periosteal bone formation, and area of bone formation increased with increasing peak periosteal strain. In these mice, the loaded tibia consistently showed lower endocortical formation surface and mineral apposition rate than the nonloaded bones at every load level. Although periosteal expansion is the most efficient means of increasing moment of inertia in adaptation to bending, a dose response increase in endocortical formation would have been predicted. Our characterization of the mouse bone formation response to increasing bending loads will be useful in the design of experiments to study the tibial adaptive response to known loads in different mouse breeds. Received: 17 February 1998 / Accepted: 9 December 1998     

Transient precursor strategy or very small biological apatite crystals?

The mechanisms of skeletal mineralization have been studied and debated for decades. Recent Raman spectroscopic identification of octacalcium phosphate-like phosphate ions and possibly amorphous calcium phosphate ions in nascent bone mineral were claimed to support a transient precursor strategy for bone apatite formation. However, this data does not refute the theory that the newest, detectable bone mineral is very small, poorly crystalline biological apatite, because non-apatitic phosphate species have previously been identified in biological apatite and detected on the surfaces of nano-sized hydroxyapatite crystals.