Community Perspectives on Social Influences on Suicide Within a Native American Reservation.

Relative to the general population, Native Americans (NA) bear a disproportionate burden of suicide-related mortality rates. NA males and females aged 15 to 24 years experience suicide rates nearly 3 times than the U.S. all races rates in this age group. Although efforts have been made to understand and reduce suicide in tribal communities, a large portion has focused on individual characteristics with less attention given to social factors that may also inform suicide. This article aims to build on a local conceptual model of NA youth suicide by examining additional potential social factors through qualitative interviews. Findings from the thematic analysis resulted in the identification of seven perceived social influences: contagion, violence and abuse, discrimination and bullying, negative expectations, spirituality, social support, and cultural strengths. Public health approaches to reduce suicide should consider local social factors that resonate with tribal communities to build resilience.

Association of sleep characteristics with suicidal ideation and suicide attempt among adults aged 50 and older with depressive symptoms in low- and middle-income countries.

OBJECTIVES: Investigate the association of sleep characteristics with suicidal ideation and suicide attempt among middle-aged and older adults with depressive symptoms in five low- and middle-income countries (LMICs). DESIGN: Cross-sectional. SETTING: China, Ghana, India, Russia, and South Africa. PARTICIPANTS: Adults aged ≥50 years with depressive symptoms from the World Health Organization (WHO) Study on Global AGEing and Adult Health (n=2,040). MEASUREMENTS: Predictors were self-reported average sleep duration for the past 2 nights (<7 hours (shorter), 7 to <9 hours (reference), ≥9 hours (longer)), sleep quality for the past 2 nights (moderate/good/very good [both nights], poor/very poor [≥1 night]), past-month insomnia symptoms (none/mild, moderate, severe/extreme), and past-day daytime sleepiness. Outcomes were past-year suicidal ideation and suicide attempt. Analyses were adjusted for age, sex, household wealth, marital status, self-rated health, cognitive performance, number of depressive symptoms, and country of residence. RESULTS: Participants with poor/very poor sleep quality ≥1 night had greater odds of suicidal ideation (vs. moderate/good/very good sleep quality both nights). Participants with moderate and severe/extreme insomnia symptoms had greater odds of suicidal ideation and suicide attempt (vs. none/mild insomnia symptoms). In moderation analyses, greater insomnia symptoms were associated with higher odds of suicidal ideation among women only and those aged 60-60 years and ≥80 years only. CONCLUSIONS: Among middle-aged and older adults with depressive symptoms in LMICs, sleep characteristics are markers of-and potential contributors to-suicidal ideation and suicide attempt, and there was evidence of moderation by age and sex. Interventions aimed at preventing suicide-related outcomes in these populations should consider the role of sleep.

How Schools Can Promote Healthy Development for Newly Arrived Immigrant and Refugee Adolescents: Research Priorities.

BACKGROUND: The US education system must find creative and effective ways to foster the healthy development of the approximately 2 million newly arrived immigrant and refugee adolescents, many of whom contend with language barriers, limited prior education, trauma, and discrimination. We identify research priorities for promoting the school success of these youth. METHODS: The study used the 4-phase priority-setting method of the Child Health and Nutrition Research Initiative. In the final stage, 132 researchers, service providers, educators, and policymakers based in the United States were asked to rate the importance of 36 research options. RESULTS: The highest priority research options (range 1 to 5) were: evaluating newcomer programs (mean = 4.44, SD = 0.55), identifying how family and community stressors affect newly arrived immigrant and refugee adolescents' functioning in school (mean = 4.40, SD = 0.56), identifying teachers' major stressors in working with this population (mean = 4.36, SD = 0.72), and identifying how to engage immigrant and refugee families in their children's education (mean = 4.35, SD = 0.62). CONCLUSION: These research priorities emphasize the generation of practical knowledge that could translate to immediate, tangible benefits for schools. Funders, schools, and researchers can use these research priorities to guide research for the highest benefit of schools and the newly arrived immigrant and refugee adolescents they serve.

Molecular profiling of oral microbiota in jawbone samples of bisphosphonate-related osteonecrosis of the jaw.

OBJECTIVE: Infection has been hypothesized as a contributing factor to bisphosphonate (BP)-related osteonecrosis of the jaw (BRONJ). The objective of this study was to determine the bacterial colonization of jawbone and identify the bacterial phylotypes associated with BRONJ. MATERIALS AND METHODS: Culture-independent 16S rRNA gene-based molecular techniques were used to determine and compare the total bacterial diversity in bone samples collected from 12 patients with cancer (six, BRONJ with history of BP; six, controls without BRONJ, no history of BP but have infection). RESULTS: Denaturing gradient gel electrophoresis profile and Dice coefficient displayed a statistically significant clustering of profiles, indicating different bacterial population in BRONJ subjects and control. The top three genera ranked among the BRONJ group were Streptococcus (29%), Eubacterium (9%), and Pseudoramibacter (8%), while in the control group were Parvimonas (17%), Streptococcus (15%), and Fusobacterium (15%). H&E sections of BRONJ bone revealed layers of bacteria along the surfaces and often are packed into the scalloped edges of the bone. CONCLUSION: This study using limited sample size indicated that the jawbone associated with BRONJ was heavily colonized by specific oral bacteria and there were apparent differences between the microbiota of BRONJ and controls.

Characterization of skin abnormalities in a mouse model of osteogenesis imperfecta using high resolution magnetic resonance imaging and Fourier transform infrared imaging spectroscopy.

Evaluation of the skin phenotype in osteogenesis imperfecta (OI) typically involves biochemical measurements, such as histologic or biochemical assessment of the collagen produced from biopsy-derived dermal fibroblasts. As an alternative, the current study utilized non-invasive magnetic resonance imaging (MRI) microscopy and optical spectroscopy to define biophysical characteristics of skin in an animal model of OI. MRI of skin harvested from control, homozygous oim/oim and heterozygous oim/+ mice demonstrated several differences in anatomic and biophysical properties. Fourier transform infrared imaging spectroscopy (FT-IRIS) was used to interpret observed MRI signal characteristics in terms of chemical composition. Differences between wild-type and OI mouse skin included the appearance of a collagen-depleted lower dermal layer containing prominent hair follicles in the oim/oim mice, accounting for 55% of skin thickness in these. The MRI magnetization transfer rate was lower by 50% in this layer as compared to the upper dermis, consistent with lower collagen content. The MRI transverse relaxation time, T2, was greater by 30% in the dermis of the oim/oim mice compared to controls, consistent with a more highly hydrated collagen network. Similarly, an FT-IRIS-defined measure of collagen integrity was 30% lower in the oim/oim mice. We conclude that characterization of phenotypic differences between the skin of OI and wild-type mice by MRI and FT-IRIS is feasible, and that these techniques provide powerful complementary approaches for the analysis of the skin phenotype in animal models of disease.

Scaffold degradation elevates the collagen content and dynamic compressive modulus in engineered articular cartilage.

OBJECTIVE: It was hypothesized that controlled, scaffold removal in engineered cartilage constructs would improve their collagen content and mechanical properties over time in culture. DESIGN: Preliminary experiments characterized the effects of agarase on cell-free agarose disks and cartilage explants. Immature bovine chondrocytes were encapsulated in agarose, cultured to day 42, and incubated with 100 units/mL agarase for 48 h. After treatment, constructs were cultured to day 91. The compressive Young's modulus and dynamic modulus of the constructs were determined every 2 weeks and immediately after agarase treatment. Post-mechanical testing, constructs were processed for biochemistry and histology. RESULTS: Agarase treatment on explants had no detrimental effect on the cartilage matrix. Treatment applied to engineered constructs on day 42 did not affect DNA or collagen content. Agarase treatment decreased tissue GAG content (via GAG loss to the media) and Young's modulus, both of which recovered to control values over time in culture. By day 91 agarase-treated constructs possessed approximately 25% more DNA, approximately 60% more collagen, and approximately 40% higher dynamic modulus compared to untreated controls. CONCLUSIONS: Scaffold degradation increased construct collagen content and dynamic mechanical properties, affirming the experimental hypothesis. The mechanism may lie in increased nutrient transport, increased space for collagen fibril formation, and cellular response to the loss of GAG with agarase treatment. The results highlight the role of the scaffold in retaining synthesized matrix during early and late tissue formation. This work also shows promise in developing an engineered tissue that may be completely free of scaffold material for clinical implantation.

Nondegradable hydrogels for the treatment of focal cartilage defects.

Nondegradable materials have long been suggested for the treatment of articular cartilage defects; however, the mechanics of the implant/tissue system necessary to ensure long-term function are unknown. The objective of this study was to explore the performance of nondegradable hydrogel implants in cartilage defects. Our hypothesis was that the structural integrity of the implant and surrounding tissue would be influenced by the compressive modulus of the material used, and that superior results would be obtained with the implantation of a more compliant material. Poly(vinyl alcohol)-poly(vinyl pyrrolidone) hydrogel implants of two different moduli were implanted into osteochondral defects in a rabbit model. Six-month postoperative histological and mechanical data were used to assess the wear and fixation of the implants. The compliant implants remained well fixed and a thin layer of soft tissue grew over the surface of the implants. However, gross deformation of the compliant implants occurred and debris was evident in surrounding bone. The stiffer implants were dislocated from their implantation site, but with no accompanying evidence of debris or implant deformation. Our hypothesis that superior results would be obtained with implantation of a more compliant material was rejected; a compromise between the wear and fixation properties dependent on modulus was found.

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.

A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS).

OBJECTIVE: The orientation of collagen molecules is an important determinant of their functionality in connective tissues. The objective of the current study is to establish a method to determine the alignment of collagen molecules in histological sections of cartilage by polarized Fourier transform infrared imaging spectroscopy (FT-IRIS), a method based on molecular vibrations. METHODS: Polarized FT-IRIS data obtained from highly oriented tendon collagen were utilized to calibrate the derived spectral parameters. The ratio of the integrated areas of the collagen amide I/II absorbances was used as an indicator of collagen orientation. These data were then applied to FT-IRIS analysis of the orientation of collagen molecules in equine articular cartilage, in equine repair cartilage after microfracture treatment, and in human osteoarthritic cartilage. Polarized light microscopy (PLM), the most frequently utilized technique to evaluate collagen fibril orientation in histological sections, was performed on picrosirius red-stained sections for comparison. RESULTS AND CONCLUSION: Thicknesses of each zone of normal equine cartilage (calculated based on differences in collagen orientation) were equivalent as determined by PLM and FT-IRIS. Comparable outcomes were obtained from the PLM and FT-IRIS analyses of repair and osteoarthritis tissues, whereby similar zonal variations in collagen orientation were apparent for the two methods. However, the PLM images of human osteoarthritic cartilage showed less obvious zonal discrimination and orientation compared to the FT-IRIS images, possibly attributable to the FT-IRIS method detecting molecular orientation changes prior to their manifestation at the microscopic level.

Distribution of collagen cross-links in normal human trabecular bone.

UNLABELLED: Infrared imaging analysis of normal human iliac crest biopsy specimens shows a characteristic spatial variation in the nonreducible:reducible collagen cross-links at trabecular surfaces, depending on the surfaces' metabolic status. INTRODUCTION: Bone is a composite material consisting of mineral, collagen, non-collagenous proteins, and lipids. Bone collagen, mainly type I, provides the scaffold on which mineral is deposited and imparts specific mechanical properties, determined in part by the amount of collagen present, its orientation and fibril diameter, and the distribution of its cross-links. MATERIALS AND METHODS: In this study, the technique of Fourier transform infrared imaging (FTIRI) was used to determine the ratio of nonreducible:reducible cross-links, in 2- to 4-microm-thick sections from human iliac crest biopsy specimens (N = 14) at trabecular surfaces as a function of surface activity (forming versus resorbing), with an approximately 6.3-mm spatial resolution. The biopsy specimens were obtained from patients devoid of any metabolic bone disease based on histomorphometric and bone densitometric parameters. RESULTS AND CONCLUSIONS: Distributions of collagen cross-links within the first 50 mm at forming trabecular surfaces demonstrated a progressive increase in the nonreducible:reducible collagen cross-link ratio, unlike in the case of resorbing surfaces, in which the collagen cross-links ratio (as defined for the purposes of the present report) was relatively constant.

Spectroscopic imaging of mineral maturation in bovine dentin.

Dentin is a useful model for the study of mineral maturation. Using Fourier Transform Infrared Imaging (FTIRI), we characterized distinct regions in developing dentin at 7- micro m spatial resolution. Mineral-to-matrix ratio and crystallinity in bovine dentin from cervical and incisal parts of 3rd-trimester fetal compared with one-year-old incisor crowns showed that virtually all maturation stages in dentin could be spectroscopically isolated and analyzed. In the fetal incisors, mantle and circumpulpal dentin presented distinct patterns of mineral maturation. Gradients in both mineral properties examined were observed at the mineralization front and at the dentino-enamel junction.

Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone.

Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) were used to characterize the mineral in bones of two different lines of Opn-deficient (Opn-/-) mice and their background-matched wild-type controls (Opn+/+). Sections of tibia and femur from 12-week-old and 16-week-old mice were evaluated with a spatial resolution between 10 microm (FTIRM) and 7 microm (FTIRI). FTIRI was used to examine 400 microm x 400 microm areas in cortical bone and trabecular bone and FTIRM examined selected 20 microm x 20 microm areas at sites within these anatomically defined areas. Despite the absence of an obvious phenotype in Opn-deficient mice, being undetectable by radiographic and histological methods, FTIRM analyses revealed that the relative amount of mineral in the more mature areas of the bone (central cortical bone) of Opn-knockout mice was significantly increased. Moreover, mineral maturity (mineral crystal size and perfection) throughout all anatomic regions of the Opn-deficient bone was significantly increased. The 2-dimensional, color-coded data (images) produced by FTIRI showed similar increases in mineral maturity in the Opn-/- bone, however, the crystallinity parameters were less sensitive, and significance was not achieved in all areas analyzed. Nonetheless, the findings of increased mineral content and increased crystal size/perfection in both lines of Opn-deficient mice at both ages are consistent with in vitro data indicating that Opn is a potent inhibitor of mineral formation and mineral crystal growth and proliferation, and also support a role for Opn in osteoclast recruitment and function.

Optimal methods for processing mineralized tissues for Fourier transform infrared microspectroscopy.

Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) are techniques utilized in the analysis of bone mineral and matrix properties in health and disease. Since the spatial arrangement of bone tissue is conserved using FTIRM and FTIRI, quantitative data can be obtained on bone mineral (hydroxyapatite) crystalline size and composition, and on matrix structure and composition at discrete anatomic locations with a spatial resolution from approximately 7 mm (FTIRI) to 10 mm (FTIRM). To section bone for FTIRM and FTIRI, it must be preserved ("fixed") to maintain its properties, and embedded in a hard supportive material. Since most of the embedding media have components that spectrally overlap the components of mineralized tissues, it is critical to define optimal embedding and fixation protocols that have the least effect on mineral and matrix spectra. In the current study, the spectra of mouse calvaria in seven different fixatives and six different commonly used embedding media were assessed by FTIRM and FTIRI. The fixatives evaluated were absolute ethanol, 70% ethanol, glycerol, formaldehyde, EM fixative, and formalin in cacodylate or phosphate-buffered saline. The embedding media tested were Araldite, Epon, JB-4, LR White, PMMA, and Spurr. Comparisons were made to FTIR spectra obtained from unprocessed ground calvaria and to spectra of cryosections of unfixed tissue, fast-frozen in polyvinyl alcohol (5% PVA). Non-aqueous fixatives and embedding in LR White, Spurr, Araldite, and PMMA had the least effect on the spectral parameters measured (mineral to matrix ratio, mineral crystallinity, and collagen maturity) compared with cryo-sectioned calvaria and non-fixed, non-embedded calvaria in KBr pellets.

BMP-6 accelerates both chondrogenesis and mineral maturation in differentiating chick limb-bud mesenchymal cell cultures.

Chick limb-bud mesenchymal cells, plated in micromass culture, differentiate in vitro to form a cartilaginous structure analogous to the epiphyseal growth plate. When inorganic phosphate, Pi, is included in the medium such that the total Pi concentration is 4 mM, apatite mineral precipitates around the "hypertrophic" chondrocytes. These hypertrophic chondrocytes are characterized by their increased expression of type X collagen, alkaline phosphatase activity, and apoptosis, as well as by the ability of their extracellular matrices to support mineral deposition. Under standard mineralizing conditions (0.8 x 10(6)cells/micromass; 4 mM Pi, 1.3 mM Ca(2+), 10% FCS, and antibiotics) mineralization does not commence until day 14-16. Based on the ability of bone morphogenic protein 6 (BMP-6) to stimulate chondrocyte maturation in other systems, 100 ng/ml BMP-6 was added to chick limb-bud mesenchymal cell cultures 2 and 5 days after plating, and the effects of this addition on mineral accretion and the characteristics of the mineral and matrix determined. Addition of BMP-6 accelerated the differentiation of the mesenchymal cells to hypertrophic chondrocytes. In the presence of BMP-6 added on both days 2 and 5, mineralization (assessed on basis of (45)Ca uptake) commenced by day 12. Fourier transform infrared imaging (FTIRI) was used to monitor the mineral content and mineral crystallinity as a function of time from day 9 to 21 in cultures with and without exogenous BMP-6. While BMP-6 accelerated the rate of mineral accretion, and the crystals that were formed in the BMP-6 cultures were initially more mature, by day 21 the crystal size distribution in experimental and control cultures were not significantly different. This study, the first to report the detailed application of FTIRI to cell cultures, indicates the importance of the extracellular matrix in the control of crystal maturation.

Trichloroethylene oxidative metabolism in plants: the trichloroethanol pathway.

Trichloroethylene (TCE) is a widespread and persistent environmental contaminant. Recently, plants, poplar trees in particular, have been investigated as a tool to remove TCE from soil and groundwater. The metabolism of TCE in plants is being investigated for two reasons: one, plant uptake and metabolism represent an important aspect of the environmental fate of the contaminant; two, metabolism pattern and metabolite identification will help assess the applicability of phytoremediation. It was previously shown that TCE metabolites in plants are similar to ones that result from cytochrome P450-mediated oxidation in mammals: trichloroethanol, trichloroacetate and dichloroacetate. Our measurements indicate that one of these metabolites, trichloroethanol, is further glycosylated in tobacco and poplar. The glycoside was detected in all tissues (roots, stems and leaves) in comparable levels, and was at least 10 fold more abundant than free trichloroethanol. The glycoside in tobacco was identified as the ss-D-glucoside of trichloroethanol by comparison of the mass spectra and the chromatographic retention time of its acetylation product to that of the synthesized standard. Trichloroethanol and its glucoside did not persist in plant tissue once plants are removed from TCE contaminated water, indicating further metabolism.

A controlled study of the effects of alendronate in a growing mouse model of osteogenesis imperfecta.

Recent studies have reported that bisphosphonates reduce fracture incidence and improve bone density in children with osteogenesis imperfecta (OI). However, questions still persist concerning the effect of these drugs on bone properties such as ultrastructure and quality, particularly in the growing patient. To address these issues, the third-generation bisphosphonate alendronate was evaluated in the growing oim/oim mouse, an animal model of moderate-to-severe OI. Alendronate was administered to 6-week-old mice during a period of active growth at a dosage of 73 microg alendronate/kg/day for the first 4 weeks and 26 microg alendronate/kg/day for the next 4 weeks. Positive treatment effects included a reduction in the number of fractures sustained by the alendronate-treated oim/oim mice compared with untreated oim/oim mice (2.1+/-2.0 vs 3.2+/-1.6 fractures per mouse), increased femoral metaphyseal density (0.111+/-0.02 vs 0.034+/-0.04 g/cm2), a tendency towards reduced tibial bowing (4.0+/-3.7 vs 6.1+/-5.8 degrees), and towards increased femoral diameter (1.22+/-0.12 vs 1.15+/-0.11 mm). Potential negative effects included a persistence of calcified cartilage in the treated oim/oim metaphyses compared with treated wildtype (+/+) (33.8+/-11.1 vs 22.1+/-10.2%), and significantly shorter femora compared with nontreated oim/oim mice (14.8+/-0.67 vs 15.3+/-0.37 mm). This preclinical study demonstrates that alendronate is effective in reducing fractures in a growing mouse model of OI, and is also an important indicator of potential positive and negative outcomes of third-generation bisphosphonate therapy in children with OI.

Spectroscopic characterization of collagen cross-links in bone.

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at approximately 1660 cm(-1) and approximately 1690 cm(-1) was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (approximately 5 microm) calcified tissue sections with a spatial resolution of approximately 7 microm.

Intradiskal electrothermal therapy: a preliminary histologic study.

OBJECTIVE: To characterize descriptively the histologic and temperature effects of intradiskal electrothermal annuloplasty on human cadaveric lumbar disks. DESIGN: In vitro histologic study. SETTING: Hospital-based soft-tissue research laboratory. CADAVERS: Six human cadaveric lumbar disks, from 5 cadavers aged 39 to 79 who died from nonspine-related causes. INTERVENTIONS: Intradiskal electrothermal therapy (IDET) by using a standard high-temperature heating protocol with the temperature of the probe gradually increased from 65 degrees C to 90 degrees C over 16.5 minutes. Disks were stained and examined by light microscopy and electron microscopy. MAIN OUTCOME MEASURES: Temperatures in outer annulus, gross macroscopic changes, and histologic damage. RESULTS: Gross inspection showed a small circumferential area of tissue alteration localized to the posterior annulus but not extending to the endplates. Light microscopy of the posterior aspect of the lumbar disks showed denaturation, shrinkage, and coalescence of annular collagen; the anterior portions, which served as internal controls, showed no evidence of damage. The endplates were structurally preserved and showed no evidence of damage. Electron microscopy showed extensive collagen disorganization, decreased quantity of collagen, collagen fibril shrinkage, and chondrocyte damage when compared with a control portion. The temperature curves showed parallel changes in temperature at the level of the probe and at the posterior portion of the disk. CONCLUSIONS: IDET raises temperatures sufficiently to induce collagen denaturation and coalescence. These histologic changes may play a substantial role in the clinical efficacy of IDET.

Physicochemical study of plasma-sprayed hydroxyapatite-coated implants in humans.

This study represents the first report of the physical and chemical changes occurring in coatings of failed hydroxyapatite (HA)-coated titanium implants obtained from a comprehensive, multicenter human dental implant study. A total of 53 retrieved samples were obtained and compared with unimplanted controls with the same manufacturer and similar manufacture dates. Forty-five retrieved implants were examined for surface characteristics and bulk composition. Implants were staged based on implantation history: stage 1 (implants retrieved between surgical placement and surgical uncovering), stage 2 (implants retrieved at surgical uncovering and evaluation), stage 3 (implants retrieved between surgical uncovering evaluation and occlusal loading), and stage 4 (implants retrieved after occlusal loading). Scanning electron microscopy showed progressive coating thinning with implantation time. At later stages, bare Ti metal was detected by energy-dispersive X-ray analysis and electron spectroscopy for chemical analysis. Increases in Ti and Al (2-7.5 atm % each) were detected at the apical ends of all stage 4 samples. In unimplanted coatings, X-ray diffraction analysis demonstrated the presence of amorphous calcium phosphate, beta-tricalcium phosphate, tetracalcium phosphate, and calcium oxide in addition to large hydroxyapatite crystals (c axis size, D002 = 429 +/- 13 A; a axis size, D300 = 402 +/- 11 A, a/c aspect ratio 0.92). The nonapatitic phases disappeared with increased implantation time, although there was a persistence of amorphous calcium phosphate. Bulk coating chemical analysis showed that Ca/P ratios for implant controls (1.81 +/- 0.01) were greater than stoichiometric HA (1.67) and decreased for implant stages 3 and 4 (1.69 +/- 0.09 and 1.67 +/- 0.09, respectively), explained by the dissolution of the non apatitic phases. Crystal sizes also changed with implantation times, being smaller than the control at all but stage 4. Fourier transform infrared analyses agreed with these results, and also indicated the accumulation of bone (protein and carbonate-apatite) in the retrieved coatings. The accumulation of bone was not stage dependent. These findings indicate that there was some biointegration with the surrounding bone, but the greatest changes occurred with the HA coating materials, their loss, and chemical change.

Homocysteine decreases chondrocyte-mediated matrix mineralization in differentiating chick limb-bud mesenchymal cell micro-mass cultures.

The differentiating chick limb-bud mesenchymal cell micro-mass culture system has been used as a model for monitoring the effects of matrix modification on cell-mediated calcification. In this study, we show that treating these micro-mass cultures with homocysteine (Hcys) impairs cartilage calcification. Cultures were treated from day 2 to day 7 with two nonphysiological concentrations of Hcys equivalent to 100x and 1000x avian serum levels (0.36 and 3.6 mmol/L), and from days 9-13 with one tenth the concentration. Mineralization assays were done at days 16, 19, and 21, and matrix and cell properties were examined between days 5 and 21. Mineral accretion, based on differential (45)Ca uptake (mineralizing minus control cultures), was significantly reduced in the high-Hcys-concentration group, and slightly reduced in the low-Hcys-concentration group. Electron microscopy at culture day 21 showed that the collagen matrix was less abundant and its banding pattern less obvious in the Hcys-treated groups than in the untreated cultures. Pyridinoline (Pyr) and deoxypyridinoline (d-Pyr) contents were not detectable in day 21 cultures with either 0.36 or 3.6 mmol/L homocysteine, whereas values in mineralizing and nonmineralizing controls ranged from 0.06 to 0.08 and 0.03 to 0.06 (moles/mole collagen) for Pyr and d-Pyr, respectively. Fourier transform infrared (FTIR) imaging also indicated a decreased content of pyridinoline cross-links. Hcys caused other matrix changes as well. Whereas at culture day 5 there was no significant difference in the number of chondrocyte nodules formed, by day 11 the proteoglycan content (measured by Alcian blue dye binding at 595 nm) was significantly reduced in both mineralizing and control cultures in the high- and low-Hcys groups. In contrast, there were no detectable differences in type X collagen and alkaline phosphatase staining in the mineralizing cultures with or without Hcys supplements. Because vital dye stains and electron microscopy studies indicated that cells in the control and experimental groups did not differ in terms of viability, the observed differences cannot be attributed to toxicity. Thus, Hcys treatment, which causes matrix disorganization, decreases the ability of the matrix to support mineralization.