Insights into the bisphosphonate holiday: a preliminary FTIRI study.

Bone composition evaluated by FTIRI analysis of iliac crest biopsies from post-menopausal women treated with alendronate for 10 years, continuously or alendronate for 5 years, followed by a 5-year alendronate-holiday, only differed with the discontinued biopsies having increased cortical crystallinity and heterogeneity of acid phosphate substitution and decreased trabecular crystallinity heterogeneity. INTRODUCTION: Bisphosphonates (BP) are the most commonly used and effective drugs to prevent fragility fractures; however, concerns exist that prolonged use may lead to adverse events. Recent recommendations suggest consideration of a BP "holiday" in individuals taking long-term BP therapy not at high risk of fracture. Data supporting or refuting this recommendation based on bone quality are limited. We hypothesized that a "holiday" of 5 years would cause no major bone compositional changes. METHODS: We analyzed the 31 available biopsies from the FLEX-Long-term Extension of FIT (Fracture Intervention Trial) using Fourier transform infrared imaging (FTIRI). Biopsies from two groups of post-menopausal women, a "Continuously treated group" (N = 16) receiving alendronate for ~ 10 years and a "Discontinued group" (N = 15), alendronate treated for 5 years taking no antiresorptive medication during the following 5 years. Iliac crest bone biopsies were provided at 10 years. RESULTS: Key FTIRI parameters, mineral-to-matrix ratio, carbonate-to-phosphate ratio, acid phosphate substitution, and collagen cross-link ratio as well as heterogeneity of these parameters were similar for Continuously treated and Discontinued groups in age-adjusted models. The Discontinued group had 2% greater cortical crystallinity (p = 0.01), 31% greater cortical acid phosphate heterogeneity (p = 0.02), and 24% lower trabecular crystallinity heterogeneity (p = 0.02). CONCLUSIONS: Discontinuation of alendronate for 5 years did not affect key FTIRI parameters, supporting the hypothesis that discontinuation would have little impact on bone composition. Modest differences were observed in three parameters that are not likely to affect bone mechanical properties. These preliminary data suggest that a 5-year BP holiday is not harmful to bone composition.

Saturation of the biological response to orthodontic forces and its effect on the rate of tooth movement.

OBJECTIVES: Investigate the expression and activity of inflammatory markers in response to different magnitudes of orthodontic forces and correlate this response with other molecular and cellular events during orthodontic tooth movement. SETTING AND SAMPLE POPULATION: CTOR Laboratory; 245 Sprague Dawley male rats. METHODS AND MATERIALS: Control, sham, and 5 different experimental groups received different magnitudes of force on the right maxillary first molar using a coil spring. In the sham group, the spring was not activated. Control group did not receive any appliance. At days 1, 3, 7, 14, and 28, the maxillae were collected for RNA and protein analysis, immunohistochemistry, and micro-CT. RESULTS: There was a linear relation between the force and the level of cytokine expression at lower magnitudes of force. Higher magnitudes of force did not increase the expression of cytokines. Activity of CCL2, CCL5, IL-1, TNF-α, RANKL, and number of osteoclasts reached a saturation point in response to higher magnitudes of force, with unchanged rate of tooth movement. CONCLUSION: After a certain magnitude of force, there is a saturation in the biological response, and higher forces do not increase inflammatory markers, osteoclasts, nor the amount of tooth movement. Therefore, higher forces to accelerate the rate of tooth movement are not justified.

Vibrational spectroscopic imaging for the evaluation of matrix and mineral chemistry.

Metabolic bone diseases manifesting fragility fractures (such as osteoporosis) are routinely diagnosed based on bone mineral density (BMD) measurements, and the effect of various therapies also evaluated based on the same outcome. Although useful, it is well recognized that this metric does not fully account for either fracture incidence or the effect of various therapies on fracture incidence, thus, the emergence of bone quality as a contributing factor in the determination of bone strength. Infrared and Raman vibrational spectroscopic techniques are particularly well-suited for the determination of bone quality as they provide quantitative and qualitative information of the mineral and organic matrix bone components, simultaneously. Through the use of microspectroscopic techniques, this information is available in a spatially resolved manner, thus, the outcomes may be easily correlated with outcomes from techniques such as histology, histomorphometry, and nanoindentation, linking metabolic status with material properties.

Chondrogenic ATDC5 cells: an optimised model for rapid and physiological matrix mineralisation.

The development of chondrogenic cell lines has led to major advances in the understanding of how chondrocyte differentiation is regulated, and has uncovered many signalling pathways and gene regulatory mechanisms required to maintain normal function. ATDC5 cells are a well established in vitro model of endochondral ossification; however, current methods are limited for mineralisation studies. In this study we demonstrate that culturing cells in the presence of ascorbic acid and 10 mM ß-glycerophosphate (ßGP) significantly increases the rate of extracellular matrix (ECM) synthesis and reduces the time required for mineral deposition to occur to 15 days of culture. Furthermore, the specific expression patterns of Col2a1 and Col10a1 are indicative of ATDC5 chondrogenic differentiation. Fourier transform-infrared spectroscopy analysis and transmission electron microscopy (TEM) showed that the mineral formed by ATDC5 cultures is similar to physiological hydroxyapatite. Additionally, we demonstrated that in cultures with ßGP, the presence of alkaline phosphatase (ALP) is required for this mineralisation to occur, further indicating that chondrogenic differentiation is required for ECM mineralisation. Together, these results demonstrate that when cultured in the presence of ascorbic acid and 10 mM ßGP, ATDC5 cells undergo chondrogenic differentiation and produce a physiological mineralised ECM from Day 15 of culture onwards. The rapid and novel method for ATDC5 culture described in this study is a major improvement compared with currently published methods and this will prove vital in the pursuit of underpinning the molecular mechanisms responsible for poor linear bone growth observed in a number of chronic diseases such as cystic fibrosis, chronic kidney disease, rheumatological conditions and inflammatory bowel disease.

Osteogenic effect of high-frequency acceleration on alveolar bone.

Mechanical stimulation contributes to the health of alveolar bone, but no therapy using the osteogenic effects of these stimuli to increase alveolar bone formation has been developed. We propose that the application of high-frequency acceleration to teeth in the absence of significant loading is osteogenic. Sprague-Dawley rats were divided among control, sham, and experimental groups. The experimental group underwent localized accelerations at different frequencies for 5 min/day on the occlusal surface of the maxillary right first molar at a very low magnitude of loading (4 µÎµ). Sham rats received a similar load in the absence of acceleration or frequency. The alveolar bone of the maxilla was evaluated by microcomputed tomography (µCT), histology, fluorescence microscopy, scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR imaging), and RT-PCR for osteogenic genes. Results demonstrate that application of high-frequency acceleration significantly increased alveolar bone formation. These effects were not restricted to the area of application, and loading could be replaced by frequency and acceleration. These studies propose a simple mechanical therapy that may play a significant role in alveolar bone formation and maintenance.

Comparable outcomes in fracture reduction and bone properties with RANKL inhibition and alendronate treatment in a mouse model of osteogenesis imperfecta.

UNLABELLED: We report a direct comparison of receptor activator of nuclear factor kappa B ligand (RANKL) inhibition (RANK-Fc) with bisphosphonate treatment (alendronate, ALN) from infancy through early adulthood in a mouse model of osteogenesis imperfecta. Both ALN and RANK-Fc decreased fracture incidence to the same degree with increases in metaphyseal bone volume via increased number of thinner trabeculae. INTRODUCTION: The potential therapeutic benefit of RANKL inhibitors in osteogenesis imperfecta (OI) is under investigation. We report a direct comparison of RANKL inhibition (RANK-Fc) with bisphosphonate treatment (ALN) from infancy through early adulthood in a model of OI, the oim/oim mouse. METHODS: Two-week-old oim/oim, oim/+, and wildtype (+/+) mice were treated with RANK-Fc 1.5 mg/kg twice per week, ALN 0.21 mg/kg/week or saline (n = 12-20 per group) for 12 weeks. RESULTS: ALN and RANK-Fc both decreased fracture incidence (9.0 ± 3.0 saline 4.4 ± 2.7 ALN, 4.3 ± 3.0 RANK-Fc fractures per mouse). Serum TRACP-5b activity decreased to 65% after 1 month in all treated mice, but increased sacrifice with RANK-Fc to 130-200% at sacrifice. Metaphyseal density was significantly increased with ALN in +/+ and oim/oim mice (p < 0.05) and tended to increase with RANK-Fc in +/+ mice. No changes in oim/oim femur biomechanical parameters occurred with treatment. Both ALN and RANK-Fc significantly increased trabecular number (3.73 ± 0.77 1/mm for oim/oim saline vs 7.93 ± 0.67 ALN and 7.34 ± 1.38 RANK-Fc) and decreased trabecular thickness (0.045 mm ± 0.003 for oim/oim saline vs 0.034 ± 0.003 ALN and 0.032 ± 0.002 RANK-Fc) and separation in all genotypes (0.28 ± 0.08 mm for oim/oim saline vs 0.12 ± 0.010 ALN and 13 ± 0.03 RANK-Fc)., with significant increase in bone volume fraction (BVF) with ALN, and a trend towards increased BVF in RANK-Fc. CONCLUSION: Treatment of oim/oim mice with either a bisphosphonate or a RANK-Fc causes similar decreases in fracture incidence with increases in metaphyseal bone volume via increased number of thinner trabeculae.

Lathyrism-induced alterations in collagen cross-links influence the mechanical properties of bone material without affecting the mineral.

In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone. Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47 day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4 weeks and ß-APN treated animals were fed additionally with ß-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2-L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively. The data of the present study indicate that ß-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p<0.05). Further, compression tests revealed a significant negative impact of ß-APN treatment on maximal force to failure and energy to failure, while stiffness was not influenced. Bone mineral density distribution (BMDD) was not altered either. At the material level, ß-APN treated rats exhibited increased Pyd/Divalent cross-link ratios in areas confined to a newly formed bone. Moreover, nano-indentation experiments showed that the E-modulus and hardness were reduced only in newly formed bone areas under the influence of ß-APN, despite a similar mineral content. In conclusion the results emphasize the pivotal role of collagen cross-links in the determination of bone quality and mechanical integrity. However, in this rat animal model of lathyrism, the coupled alterations of tissue structural properties make it difficult to weigh the contribution of the anatomically confined material changes to the overall mechanical performance of whole bone. Interestingly, the collagen cross-link ratio in bone forming areas had the same profile as seen in actively bone forming trabecular surfaces in human iliac crest biopsies of osteoporotic patients.

MicroCT morphometry analysis of mouse cancellous bone: intra- and inter-system reproducibility.

The agreement between measurements and the relative performance reproducibility among different microcomputed tomography (microCT) systems, especially at voxel sizes close to the limit of the instruments, is not known. To compare this reproducibility 3D morphometric analyses of mouse cancellous bone from distal femoral epiphyses were performed using three different ex vivo microCT systems: GE eXplore Locus SP, Scanco µCT35 and Skyscan 1172. Scans were completed in triplicate at 12 µm and 8 µm voxel sizes and morphometry measurements, from which relative values and dependence on voxel size were examined. Global and individual visually assessed thresholds were compared. Variability from repeated scans at 12 µm voxel size was also examined. Bone volume fraction and trabecular separation values were similar, while values for relative bone surface, trabecular thickness and number varied significantly across the three systems. The greatest differences were measured in trabecular thickness (up to 236%) and number (up to 218%). The relative dependence of measurements on voxel size was highly variable for the trabecular number (from 0% to 20% relative difference between measurements from 12 µm and 8 µm voxel size scans, depending on the system). The intra-system reproducibility of all trabecular measurements was also highly variable across the systems and improved for BV/TV in all the systems when a smaller voxel size was used. It improved using a smaller voxel size in all the other parameters examined for the Scanco system, but not consistently so for the GE or the Skyscan system. Our results indicate trabecular morphometry measurements should not be directly compared across microCT systems. In addition, the conditions, including voxel size, for trabecular morphometry studies in mouse bone should be chosen based on the specific microCT system and the measurements of main interest.

Aging and bone.

Bones provide mechanical and protective function, while also serving as housing for marrow and a site for regulation of calcium ion homeostasis. The properties of bones do not remain constant with age; rather, they change throughout life, in some cases improving in function, but in others, function deteriorates. Here we review the modifications in the mechanical function and shape of bones, the bone cells, the matrix they produce, and the mineral that is deposited on this matrix, while presenting recent theories about the factors leading to these changes.

The effect of lead on bone mineral properties from female adult C57/BL6 mice.

Lead toxicity is a significant problem in the U.S. with elevated blood lead levels being highest among very young children and older adults >50 years old. Bone is the major reservoir of body lead, accounting for 75% in children and 90% in adults. Very little is known about the effect of lead on bone mineral properties in adults. We investigated the effect of lead on the femora from adult, 6 month old female C57/BL6 mice who were administered lead in the drinking water (250 ppm, blood lead 33 µg/dL) for 4 months. Bone mineral properties were examined using Fourier Transform Infrared Microscopy (FTIRM), quantitative microcomputed tomography (microCT) and whole bone mechanical testing. Lead significantly decreased the bone mineral density in the cortical and proximal cancellous bone and increased the marrow area in the cortical bone with microCT. Whole bone three-point bending showed a trend of decreased maximum and failure moments in the lead treated bones compared to controls. Lead significantly decreased the mineral/matrix ratio, collagen maturity and crystallinity in the trabecular bone as measured by FTIRM. In the cortical bone lead significantly decreased collagen maturity and bone crystal size by FTIRM. In contrast to cell culture studies, lead significantly increased serum osteocalcin levels. Lead also significantly increased the bone formation and resorption markers suggesting increased bone turnover. These data show that lead increases bone turnover resulting in weaker cortical bone in adult female mice and suggest that lead may exacerbate bone loss and osteoporosis in the elderly.

Different forms of DMP1 play distinct roles in mineralization.

Dentin matrix protein-1 (DMP1) is a major synthetic product of hypertrophic chondrocytes and osteocytes. Previous in vitro studies showed full-length DMP1 inhibits hydroxyapatite (HA) formation and growth, while its N-terminal fragment (37K) promotes HA formation. Since there are 3 fragments within the mineralized tissues [N-terminal, C-terminal (57K), and a chondroitin-sulfate-linked N-terminal fragment (DMP1-PG)], we predicted that each would have a distinct effect on mineralization related to its interaction with HA. In a gelatin-gel system, 37K and 57K fragments were both promoters of HA formation and growth; DMP1-PG was an inhibitor. The secondary structures of the 3 fragments and the full-length protein in the presence and absence of Ca2+ and HA determined by FTIR showed that the full-length protein undergoes slight conformational changes on binding to HA, while 37K, 57K, and DMP1-PG do not change conformation. These findings indicate that distinct forms of DMP1 may work collectively in controlling the mineralization process.

Influence of bone-derived matrices on generation of bone in an ectopic rat model.

Most bone regeneration experimental models that test bone-derived matrices take place in conjunction with the native bone. Here, we compared the relative effectiveness of bone matrix components on bone-marrow-directed osteogenesis in an ectopic model. Cortical bone cylinders consisted of diaphysis of DA rat femurs. They were either demineralized (DBM), deproteinized (HABM), or nontreated (MBM). Fresh bone marrow was placed into cylinders and implanted at subcutaneous thoracic sites of 2-month-old DA rats. At designated times the cylinders were surgically removed from the animals. Microradiographs of DBM and histology of DBM and MBM cylinders demonstrated progressive increase in mineralized bone volume and its trabecular configuration. Bone filled the inner volume of DBM and MBM cylinders within 4 weeks, while in HABM cylinders mostly granulation tissue developed. In the DBM cylinders cartilage deposited within 10 days, while in the MBM cylinders bone was directly deposited. As early as day 3 after marrow transplantation, marrow cells interacting with DBM increased significantly the genes that express the cartilage and the bone phenotype. In conclusion, organic components of bone are needed for marrow-directed osteogenesis.

Spectroscopic markers of bone quality in alendronate-treated postmenopausal women.

UNLABELLED: Comparison of infrared spectroscopic images of sections from biopsies of placebo-treated post-menopausal women and women treated for 3 years with 10 mg/day alendronate demonstrated significant increases in cortical bone mineral content, no alterations in other spectroscopic markers of "bone quality," but a decrease in tissue heterogeneity. METHODS: The material properties of thick sections from iliac crest biopsies of seven alendronate-treated women were compared to those from ten comparably aged post-menopausal women without bone disease, using infrared spectroscopic imaging at approximately 7 microm spatial resolution. Parameters evaluated were mineral/matrix ratio, crystallinity, carbonate/amide I ratio, and collagen maturity. The line widths at half maximum of the pixel histograms for each parameter were used as measures of heterogeneity. RESULTS: The mineral content (mineral/matrix ratio) in the cortical bone of the treated women's biopsies was higher than that in the untreated control women. Crystallinity, carbonate/protein, and collagen maturity indices were not significantly altered; however, the pixel distribution was significantly narrowed for all cortical and trabecular parameters with the exception of collagen maturity in the alendronate treatment group. CONCLUSIONS: The increases in mineral density and decreased fracture risk associated with bisphosphonate treatment may be counterbalanced by a decrease in tissue heterogeneity, which could impair tissue mechanical properties. These consistent data suggest that alendronate treatment, while increasing the bone mass, decreases the tissue heterogeneity.

Maturational changes in dentin mineral properties.

In this study the changes in properties of the maturing mantle and circumpulpal dentin were quantitatively analyzed. Sections from six fetal bovine undecalcified incisors were used. Regions of mantle and circumpulpal dentin of sequential maturation stages were identified on spectroscopic images acquired by Fourier Transform Infrared Imaging. Spectroscopic parameters corresponding to mineral properties at these stages were analyzed and reported as a function of distance from the cervix of the incisor, the latter representing tissue age. Mineral parameters were correlated with distance from the cervix. Values of these parameters in mantle and circumpulpal dentin were compared. A multi-phasic pattern of changes was found for all the parameters examined, with most of the alterations occurring in the initial maturation period. The patterns of temporal variation in mantle and circumpulpal dentin mineral properties show distinct developmental stages and were not identical for the two dentin compartments. The study showed that mineral maturation in dentin is not a linear process and that mantle dentin is developmentally distinct from circumpulpal dentin, presenting at certain stages different physicochemical events during the maturation of the tissue.

Comparison of mineral quality and quantity in iliac crest biopsies from high- and low-turnover osteoporosis: an FT-IR microspectroscopic investigation.

Fourier-transform infrared microspectroscopy (FTIRM) allows analysis of mineral content, mineral crystal maturity and mineral composition at approximately 10-micron spatial resolution. Previous FTIRM analyses comparing 4-micron thick sections from non-decalcified iliac crest biopsies from women with post-menopausal osteoporosis, as contrasted with iliac crest tissue from individuals without evidence of metabolic bone disease, demonstrated significant differences in average mineral content (decreased in osteoporosis) and mineral crystal size/perfection (increased in osteoporosis). More importantly, these parameters, which vary throughout the tissue in relation to the tissue age in healthy bone, showed no such variation in bone biopsies from patients with osteoporosis. The present study compares the spatial and temporal variation in mineral quantity and properties in trabecular bone in high- and low-turnover osteoporosis. Specifically, six biopsies from women (n=5) and one man with high-turnover osteoporosis (age range 39-77) and four women and two men with low turnover osteoporosis (age range 37-63) were compared to ten "normal" biopsies from three men and seven woman (age range: 27-69). "High turnover" was defined as the presence of increased resorptive surface, higher than normal numbers of osteoclasts and greater than or equal to normal osteoblastic activity. "Low turnover" was defined as lower than normal resorptive surface, decreased osteoclast number and less than normal osteoblastic activity. Comparing variations in FTIR-derived values for each of the parameters measured at the surfaces of the trabecular bone to the maximum value observed in multiple trabeculae from each person, the high-turnover samples showed little change in the mineral: matrix ratio, carbonate: amide I ratio, crystallinity and acid phosphate content. The low-turnover samples also showed little change in these parameters, but in contrast to the high-turnover samples, the low-turnover samples showed a slight increase in these parameters, indicative of retarded, but existent resorption and formation. These data indicate that FTIR microspectroscopy can provide quantitative information on mineral changes in osteoporosis that are consistent with proposed mechanisms of bone loss.

Importance of phosphorylation for osteopontin regulation of biomineralization.

Previous in vitro and in vivo studies demonstrated that osteopontin (OPN) is an inhibitor of the formation and growth of hydroxyapatite (HA) and other biominerals. The present study tests the hypotheses that the interaction of OPN with HA is determined by the extent of protein phosphorylation and that this interaction regulates the mineralization process. Bone OPN as previously reported inhibited HA formation and HA-seeded growth in a gelatin-gel system. A transglutaminase-linked OPN polymer had similar effects. Recombinant, nonphosphorylated OPN and chemically dephosphorylated OPN, had no effect on HA formation or growth in this system. In contrast, highly phosphorylated milk OPN (mOPN) promoted HA formation. The mOPN stabilized the conversion of amorphous calcium phosphate (a non-crystalline constituent of milk) to HA, whereas bone OPN had a lesser effect on this conversion. Mixtures of OPN and osteocalcin known to form a complex in vitro, unexpectedly promoted HA formation. To test the hypothesis that small alterations in protein conformation caused by phosphorylation account for the differences in the observed ability of OPN to interact with HA, the conformation of bone OPN and mOPN in the presence and absence of crystalline HA was determined by attenuated total reflection (ATR) infrared (IR) spectroscopy. Both proteins exhibited a predominantly random coil structure, which was unaffected by the addition of Ca(2+). Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of beta-sheet (few percent) in mOPN. These data taken together suggest that the phosphorylation of OPN is an important factor in regulating the OPN-mediated mineralization process.

Overexpression of IGF-binding protein 5 alters mineral and matrix properties in mouse femora: an infrared imaging study.

The anabolic effects of insulin-like growth factors (IGFs) are modulated by a family of IGF-binding proteins (IGFBPs). Among the six known IGFBPs, IGFBP-5 is considered to play a role in bone formation. To investigate the effects of IGFBP-5 on bone mineral and matrix properties, femurs from transgenic mice overexpressing IGFBP-5 under the control of the osteocalcin promoter were evaluated by Fourier Transform Infrared Imaging (FTIRI). Analyses were done at the time of maximal osteocalcin expression (5 weeks). The spectroscopic parameters monitored were mineral-to-matrix ratio (indicative of the relative amount of mineral present), mineral crystallinity (index of the mineral crystal size and perfection) and collagen maturity (reflecting the ratio of non-reducible and reducible collagen cross-links). Multiple fields were selected for each femur, ranging from epiphysis to diaphysis. Previously, we showed that these transgenic mice display decreased osteoblastic function and osteopenia. In the present work, FTIRI showed that transgenic mice as compared to wild types have a different pattern of bone mineralization and matrix maturation. Specifically, cortical bone, primary spongiosa, and secondary ossification centers had lower values for mineral-to-matrix ratio and collagen maturity. Differences were not statistically significant in all cases although the trends were consistent. The mineral crystallinity did not vary significantly between the two groups, implying that the crystal maturation of mineral was not affected by IGFBP-5 overexpression. This study demonstrates that femurs from transgenic mice over expressing IGFBP-5 under the control of the osteocalcin promoter have modest alterations in mineral and matrix distribution, consistent with a role of IGF in osteoblast maturation.

Diffusion systems for evaluation of biomineralization.

A variety of in vitro study methods have been used to elucidate the roles of matrix molecules in biomineralization processes. Among these, gel diffusion-precipitation studies have proved to be an effective tool. This methodology is uniquely capable of characterizing the effects of matrix molecules on mineralization while only using very small quantities of material. Furthermore, gel methods have been extended for use as a mineralization assay system to characterize modified matrix molecules and synthetic analogues. Here we discuss the advantages and limitations of gelatin, agar, agarose, and other systems for studying the mechanisms of biomineralization.

Thermal and chemical modification of titanium-aluminum-vanadium implant materials: effects on surface properties, glycoprotein adsorption, and MG63 cell attachment.

The microstructure, chemical composition and wettability of thermally and chemically modified Ti-6Al-4V alloy disks were characterized and correlated with the degree of radiolabeled fibronectin-alloy surface adsorption and subsequent adhesion of osteoblast-like cells. Heating either in pure oxygen or atmosphere (atm) resulted in an enrichment of Al and V within the surface oxide. Heating (oxygen/atm) and peroxide treatment both followed by butanol treatment resulted in a reduction in content of V, but not in Al. Heating (oxygen/atm) or peroxide treatment resulted in a thicker oxide layer and a more hydrophilic surface when compared with passivated controls. Post-treatment with butanol, however, resulted in less hydrophilic surfaces than heating or peroxide treatment alone. The greatest increases in the adsorption of radiolabeled fibronectin following treatment were observed with peroxide/butanol-treated samples followed by peroxide/butanol and heat/butanol, although binding was only increased by 20-40% compared to untreated controls. These experiments with radiolabeled fibronectin indicate that enhanced adsorption of the glycoprotein was more highly correlated with changes in chemical composition, reflected in a reduction in V content and decrease in the V/Al ratio, than with changes in wettability. Despite promoting only a modest elevation in fibronectin adsorption, the treatment of disks with heat or heat/butanol induced a several-fold increase in the attachment of MG63 cells promoted by a nonadhesive concentration of fibronectin that was used to coat the pretreated disks compared to uncoated disks. Therefore, results obtained with these modifications of surface properties indicate that an increase in the absolute content of Al and/or V (heat), and/or in the Al/V ratio (with little change in hydrophilicity; heat+butanol) is correlated with an increase in the fibronectin-promoted adhesion of an osteoblast-like cell line. It would also appear that the thermal treatment-induced enhancement of cell adhesion in the presence of this integrin-binding protein is due to its increased biological activity, rather than a mass effect alone, that appear to be associated with changes in chemical composition of the metallic surface. Future studies will investigate the influence of the surface chemical composition of various implantable alloys on protein adsorption and receptor-mediated cell adhesion. In addition, by altering the properties of bound osteogenic protein enhancing exposure to cell integrin binding domains, it may be possible to develop implant surfaces which enhance the attachment, adhesion and developmental response of osteoblast precursors leading to accelerated osseointegration.