Link Between Clinical Predictors of Heterotopic Ossification and Histological Analysis in Combat-Injured Service Members.

BACKGROUND: Heterotopic ossification (HO) is a debilitating condition that occurs following traumatic injury and may restrict range of motion and delay rehabilitation. The timing and efficacy of surgical resection have varied widely, and there is a gap in knowledge between clinical predictors of HO recurrence and histological analysis. METHODS: Thirty-three service members seen at Walter Reed National Military Medical Center for symptomatic HO were enrolled in an institutional review board-approved study. Participants took oxytetracycline on four scheduled days prior to HO resection to determine the mineral apposition rate (bone growth rate). RESULTS: Detailed histological analyses included scanning electron microscopy with backscattered electron imaging and light microscopy. Data indicated that the mineral apposition rate of trauma-induced HO was approximately 1.7 µm/day at the time of operative intervention, which was 1.7 times higher than the rate in non-pathological human bone. The mineral apposition rate and postoperative alkaline phosphatase values were demonstrated to be positively and significantly related (ρ = 0.509, p = 0.026, n = 19). When the analysis was limited to patients with no more than a two-year period from injury to excision (thereby removing outliers who had a longer time period than their counterparts) and traumatic brain injury and nonsteroidal anti-inflammatory drugs (known correlates with HO development) were controlled for in the statistical analysis, the mineral apposition rate and recurrence severity were significantly related (ρ = -0.572, p = 0.041, n = 11). CONCLUSIONS: Data demonstrated a link between benchtop research and bedside care, with the mineral apposition rate elevated in patients with HO and correlated with recurrence severity; however, a larger sample size and more clinical factors are needed to refine this model. LEVEL OF EVIDENCE: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Development of a broad spectrum polymer-released antimicrobial coating for the prevention of resistant strain bacterial infections.

More than 400,000 primary hip and knee replacement surgeries are performed each year in the United States. From these procedures, approximately 0.5-3% will become infected and when considering revision surgeries, this rate has been found to increase significantly. Antibiotic-resistant bacterial infections are a growing problem in patient care. This in vitro research investigated the antimicrobial potential of the polymer released, broad spectrum, Cationic Steroidal Antimicrobial-13 (CSA-13) for challenges against 5 × 10(8) colony forming units (CFU) of methicillin-resistant Staphylococcus aureus (MRSA). It was hypothesized that a weight-to-weight (w/w) concentration of 18% CSA-13 in silicone would exhibit potent bactericidal potential when used as a controlled release device coating. When incorporated into a polymeric device coating, the 18% (w/w) broad-spectrum polymer released CSA-13 antimicrobial eliminated 5 × 10(8) CFU of MRSA within 8 h. In the future, these results will be utilized to develop a sheep model to assess CSA-13 for the prevention of perioperative device-related infections in vivo.

Examining the influence of short-term implantation on oxidative degradation in retrieved highly crosslinked polyethylene tibial components.

Concerns remain regarding the oxidative resistance of highly crosslinked polyethylene (PE). The study investigated the in vivo performance of Durasul highly crosslinked PE by comparing the oxidation index, density, and percent crystallinity in the weightbearing and nonweightbearing region of retrieved components with unused time zero tibial components. Retrieved and unused Sulene conventional PE tibial components were examined for comparison and the effects of shelf age, in vivo duration, and ex vivo duration were also investigated. The oxidation index was not significantly different between unused time zero and retrieved Durasul PE components. Regression analysis data supported these findings in that neither shelf age, in vivo duration, nor ex vivo duration was a significant predictor of oxidation index in the retrieved Durasul PE components. In contrast, the retrieved conventional PE components had significantly greater oxidation index, density, and percent crystallinity compared with unused time zero PE components. Regression data suggested that in vivo and ex vivo duration, but not shelf aging, influenced the changes observed in the conventional PE components. These data also showed that in vivo loading did not significantly affect the oxidation index, density, or percent crystallinity in either the retrieved Durasul or conventional PE tibial components. This investigation demonstrates that changes in oxidation index, density, and percent crystallinity of retrieved Durasul PE components after short-term in vivo durations are likely not a clinical concern. These data should be used as a benchmark to compare with future studies examining the long-term oxidative resistance of Durasul highly crosslinked PE tibial components.

Surface damage analysis of retrieved highly crosslinked polyethylene tibial components after short-term implantation.

The use of highly crosslinked polyethylene (PE) in the knee remains controversial, because of reduced fatigue fracture properties of the material. The current study investigated postmelt surface damage as well as potential contributors to this damage in retrieved highly crosslinked PE tibial components, after short-term in vivo durations. Retrieved conventional PE tibial components were examined for comparison, as well as unused time zero highly crosslinked and conventional PE tibial components for inherent manufacturing surface characterization. Predominant surface damage modes on highly crosslinked PE components were machine mark loss and abrasion, while conventional PE components primarily had machine mark loss, abrasion, and delamination. In vivo duration, PE thickness, and conformity of the design were significant predictors of surface damage on retrieved conventional PE components. Donor weight and the conformity of the design were significant predictors of surface damage on retrieved highly crosslinked PE components. This retrieval data on highly crosslinked PE tibial components suggest that in vivo wear occurred, observed as postmelt surface damage. The highly crosslinked Durasul material examined in this retrieval study appeared to outperform the conventional PE components made from 4150 resin, ram-extruded and gamma-sterilized in air, but not the conventional components made from 1020 resin, compression molding and gamma sterilization in nitrogen. Early retrieval data of highly crosslinked PE tibial components are important to serve as a benchmark to be compared with future longer-term retrieval studies investigating whether surface damage translates to clinically relevant particulate wear debris generation and PE clinical performance.

Relationship between bone ingrowth, mineral apposition rate, and osteoblast activity.

To better understand skeletal attachment of porous coated total hip and knee implants over time, this study investigated the dynamics of osteoblast populations at the interface of porous coated implants in a weight-bearing ovine model. The relationship between cancellous bone ingrowth, mineral apposition rate (MAR), and osteoblast activity indicators such as osteoblast area, relative osteoblast number, osteoid width, and osteoid area (O.Ar.) were investigated. The data demonstrated that the percent O.Ar. was a marginally significant predictor of bone ingrowth and MAR over time, suggesting that the amount of osteoid present influenced bone ingrowth and MAR in the porous coated implants. The data also demonstrated that all osteoblast activity indicators were significantly greater in the porous coated region compared to the host bone region, while controlling for in situ time (p < 0.05). This may have been due to the trauma of implantation or the influence of the implant load on the bone tissue promoting a regional acceleratory phenomenon. The localized response suggests that specific therapies may be developed to affect the physiology of osteoblasts at the interface of implants, which may allow for improve skeletal attachment of biomaterials and clinical outcomes of cementless joint replacements.

Levocetirizine: the allergist's arsenal grows larger.

Antihistamines are the cornerstone of treatment for many allergic diseases, such as allergic rhinitis and chronic urticaria. Since the discovery of their beneficial effects in the 1940s, scientists have found molecules with greater selectivity to block specific histamine receptors without some of the detrimental side effects that are seen if antihistamines cross the blood-brain barrier. Levocetirizine is the active enantiomer of cetirizine and a selective H(1)-histamine blocker. It exhibits many favourable characteristics of an ideal antihistamine, both pharmacodynamically and pharmacokinetically, including high bioavailability, rapid onset of action, limited distribution and low degree of metabolism. Furthermore, clinical trials indicate that it is safe and effective for the treatment of allergic rhinitis and chronic urticaria with a minimal amount of untoward effects.

Immunomodulation: the future of allergy and asthma treatment.

PURPOSE OF REVIEW: As the prevalence of asthma and allergic disease increases around the world, it is clear that more effective therapies and disease-modifying agents are needed. Treatment for allergic disease is evolving with an increase in understanding of the etiology. RECENT FINDINGS: The first immunomodulatory treatment was recently approved for use in the United States when the Food and Drug Administration approved the use of a humanized monoclonal anti-IgE antibody in patients with allergic asthma. Another strategy that has proved effective in a murine model is the downregulation of the whole immune system by targeting adhesion molecules, which has been evaluated in a recent human trial. Other strategies for the treatment of allergic diseases concentrate on refocusing the immune system away from an allergic-type response. These include the use of targeted therapies towards specific cytokines, cytokine receptors or chemokine receptors, and the use of specific bacterial DNA sequences (unmethylated cytosine-guanine dinucleotides). Finally, attention is being focused on possible therapies that may tilt the immune response to a non-allergic response by interfering with signaling molecule pathways. SUMMARY: Immunomodulation will play a key role in future therapies for allergic disease. These treatment modalities may not only treat allergic disease, but also be beneficial in reducing the morbidity and mortality for which it is responsible.

Modeling and remodeling in a developing artiodactyl calcaneus: a model for evaluating Frost's Mechanostat hypothesis and its corollaries.

The artiodactyl (mule deer) calcaneus was examined for structural and material features that represent regional differences in cortical bone modeling and remodeling activities. Cortical thickness, resorption and formation surfaces, mineral content (percent ash), and microstructure were quantified between and within skeletally immature and mature bones. These features were examined to see if they are consistent with predictions of Frost's Mechanostat paradigm of mechanically induced bone adaptation in a maturing "tension/compression" bone (Frost, 1990a,b, Anat Rec 226:403-413, 414-422). Consistent with Frost's hypothesis that surface modeling activities differ between the "compression" (cranial) and "tension" (caudal) cortices, the elliptical cross-section of the calcaneal diaphysis becomes more elongated in the direction of bending as a result of preferential (> 95%) increase in thickness of the compression cortex. Regional differences in mineral content and population densities of new remodeling events (NREs = resorption spaces plus newly forming secondary osteons) support Frost's hypothesis that intracortical remodeling activities differ between the opposing cortices: 1.) in immature and mature bones, the compression cortex had attained a level of mineralization averaging 8.9 and 6.8% greater (P < 0.001), respectively, than that of the tension cortex, and 2.) there are on average 350 to 400% greater population densities of NREs in the tension cortices of both age groups (P < 0.0003). No significant differences in cortical thickness, mineral content, porosity, or NREs were found between medial and lateral cortices of the skeletally mature bones, suggesting that no modeling or remodeling differences exist along a theoretical neutral axis. However, in mature bones these cortices differed considerably in secondary osteon cross-sectional area and population density. Consistent with Frost's hypothesis, remodeling in the compression cortex produced bone with microstructural organization that differs from the tension cortex. However, the increased remodeling activity of the tension cortex does not appear to be related to a postulated low-strain environment. Although most findings are consistent with predictions of Frost's Mechanostat paradigm, there are several notable inconsistencies. Additional studies are needed to elucidate the nature of the mechanisms that govern the modeling and remodeling activities that produce and maintain normal bone. It is proposed that the artiodactyl calcaneus will provide a useful experimental model for these studies.

The contribution of cortical and cancellous bone to dual-energy X-ray absorptiometry measurements in the female proximal femur.

Dual-energy X-ray absorptiometry (DXA) is the most common method for determining bone mineral density (BMD) in the proximal femur. However, there remain questions concerning the contribution of cortical and cancellous bone to this technology in the proximal femur. The purpose of this investigation was to identify structural and compositional characteristics of human bone in the proximal femur that significantly influence DXA BMD measurements. Twenty-four femora were obtained at autopsy from Caucasian females ranging in age from 17 to 92 years (mean +/- SD, 61 +/- 25 years). DXA scans were performed on each specimen with a Hologic QDR-2000 densitometer. Direct measurements were determined from proximal femoral sections for cancellous bone (volume fraction, ash fraction, cancellous cross-sectional area and percent cancellous cross-sectional area), cortical bone (thickness, ash fraction, porosity, cortical cross-sectional area and percent cortical cross-sectional area) and anteroposterior thickness. These parameters were compared with the associated DXA measurements by means of simple and multiple regressions. Cancellous volume fraction was the best predictor of variability of DXA measurements for both the neck and trochanter, with an R2 of 0.87 and 0.76, respectively (p < 0.0001). There was only a minor influence of cortical factors such as thickness (neck and trochanter R2 = 0.51 and 0.42, respectively, p < 0.001) and trochanteric cross-sectional area (R2 = 0.21, p < 0.05). Although the accuracy for determining specific components of the proximal femur was low, the DXA BMD measurement was a strong predictor of cancellous bone factors, but not cortical bone factors that have been shown to change significantly with age.

Evidence of a hypermineralised calcified fibrocartilage on the human femoral neck and lesser trochanter.

Femoral neck fractures are a major cause of morbidity and mortality in elderly humans. In addition to the age-related loss of cancellous bone, changes to the microstructure and morphology of the metaphyseal cortex may be a contributing factor in osteoporotic hip fractures. Recent investigations have identified a hypermineralised tissue on the neck of the femur and trochanteric region that increases in fractional area with advancing age in both males (Boyce & Bloebaum, 1993) and females (Vajda & Bloebaum, 1999). The aim of this study was to determine if the hypermineralised tissue previously observed on the proximal femur is calcified fibrocartilage. Regional variations in the fractional area of hypermineralised tissue, cortical bone, and porosity of the cortical bone along the neck of the femur and lesser trochanter were also quantified. Comparison of back scattered electron and light microscope images of the same area show that regions of hypermineralised tissue correlate with the regions of calcified fibrocartilage from tendon and capsular insertions. The hypermineralised tissue and calcified fibrocartilage had similar morphological features such as the interdigitations of the calcified fibrocartilage into the bone, lacunar spaces, and distinctly shaped pores adjacent to the 2 tissues. Regions of the neck that did not contain insertions were covered with periosteum. There were no regional differences (P > 0.05) on the superior and inferior femoral neck in terms of the percentage area of hypermineralised calcified fibrocartilage, cortical bone, or cortical bone porosity. The lesser trochanter exhibited regional differences in the fractional area of hypermineralised calcified fibrocartilage (P = 0.007) and cortical bone (P = 0.007) but not porosity of the cortical bone (P > 0.05). The effects of calcified fibrocartilage on femoral neck periosteal expansion, repair, and mechanics are unknown, but may play a role in osteoporotic fractures and intracapsular fracture healing.

Possible explanation for the white band artifact seen in clinically retrieved polyethylene tibial components.

Studies have focused attention on the appearance of a subsurface white band in clinically retrieved polyethylene components and the possible contribution of this phenomenon to early polyethylene delamination. Unconsolidated polyethylene particles and oxidation have been suggested as possible reasons for the appearance of the white band. Calcium stearate and other additives used in processing ultra-high molecular weight polyethylene may also contribute to formation of the white band. A quantitative investigation was conducted on 11 retrieved tibial components that exhibited a subsurface white band to determine whether the amount of calcium stearate particles and additives were greater in the white band region when compared with the mid-portion of the same section of polyethylene. Calcium stearate particles and other additives were quantified using backscattered electron imaging with correlated elemental analysis. The particles were identified based on morphology and elemental patterns similar to reference calcium stearate particles and known additives. Significantly more (p < 0. 0001) calcium stearate particles and additives were present in the white band region (4578 +/- 418 particles/mm(2); mean +/- standard error) than the mid-portion region (1250 +/- 147 particles/mm(2)) of the sectioned tibial inserts. The percent area occupied by calcium stearate particles and additives was five times higher (p < 0.0001) within the white band region (0.81 +/- 0.10%) than the mid-portion region (0.16 +/- 0.03%). The increased presence of calcium stearate and other additives in the white band region suggests that they may play a role in the formation of the white band. In future investigations it may be important to consider how calcium stearate and other additives in polyethylene resins affect white band formation and the possible contribution to crazing, early delamination, and osteolysis in total joint replacement.

Age-related cancellous bone loss in the proximal femur of caucasian females.

The purpose of this investigation was to directly define the age-related intrafemoral variations in cancellous bone density, bone mineralization and rate of bone loss in a cadaveric population of Caucasian female femoral necks and trochanters. Forty-three Caucasian female femora were obtained and divided into premenopausal, postmenopausal and elderly age groups. The neck and trochanter were removed, and cores of cancellous bone were taken from the superior, middle and inferior regions; volume fraction and ash fraction were determined for each core. The cancellous bone volume fraction of the neck was significantly greater than that of the trochanter, as was that of the inferior region of the neck compared with the superior and middle regions at all age groups (p<0.05). The mean neck/trochanter and neck inferior/superior volume fraction ratios did not change with age; however, the variance increased with age (p<0.001). This increasing variability with age suggests that there may be a subpopulation of individuals within the elderly Caucasian population with a significantly different intrafemoral bone density distribution than was present prior to menopause. This study identified no mineralization changes with age in the cancellous bone of the proximal femur (p>0.05). The influence of increased neck/trochanter and neck inferior/superior ratios on femoral neck integrity and fracture prediction is of interest and requires further investigation.

Thermal effects of the electron beam and implications of surface damage in the analysis of bone tissue.

Electron beam interactions with specimens in the scanning electron microscope (SEM) can lead to increased surface temperatures and damage. These changes may have significant consequences in the analysis of bone tissue. An investigation was performed to measure the surface temperature changes associated with the electron beam on a thermocouple with systematic variations in operating conditions. Probe currents, magnifications, and accelerating voltages were incrementally adjusted to measure the temperature changes and to make assessments for determining optimal operating conditions for the SEM in future analyses of bone tissue. Results from this study suggest that thermal effects were minimal at lower accelerating voltages (< 20 kV), lower probe currents (< 10 nA), and lower magnifications, but surface damage may still occur during the analysis of bone tissue.

Elemental and morphological identification of third-body particulate and calcium stearate inclusions in polyethylene components.

Third-body particulate such as human bone chips, hydroxyapatite, and bone cement are considered contributing factors in accelerated wear in total joint replacement. Particulate wear debris is now considered the major contributing factor in aseptic loosening of total joint replacements. The ability to distinguish between different third-body particulate is necessary to better understand wear mechanisms when conducting implant retrieval analysis. The objective of this investigation is to demonstrate that backscattered electron imaging with correlated energy dispersive X-ray analysis can accurately identify third-body particulate in retrieved polyethylene components. It is important that this technique can also distinguish between third-body particulate and normal inclusions in the polyethylene such as calcium stearate, based on the distinct morphology and elemental composition of each material. Therefore, the ability to distinguish third-body particulate from calcium stearate inclusions is essential in gaining a better understanding of the contributing factors associated with coating separation and accelerated wear observed in clinically retrieved polyethylene components.

Loading conditions and cortical bone construction of an artiodactyl calcaneus.

Customary nonuniform distributions of physiological bone strains are thought to evoke heterogeneous material adaptation in diaphyseal cortices of some limb bones. Recent studies of artiodactyl calcanei have suggested that the regional prevalence of specific mechanical strain features such as mode and magnitude correlate with specific variations in cortical bone ultrastructure, microstructure and mineralization. These data are also consistent with predictions of current algorithms of mechanically induced bone adaptation. However, detailed characterization of the customary functional strain environment of these bones is needed to understand better the mechanisms of these adaptations. An in vitro loading method and rosette strain gauges were used to record principal strains, maximum shear strains and principal strain angles at multiple locations on ten calcanei of adult male mule deer (Odocoileus hemionus hemionus). Each hind limb was fixed in an apparatus to mimic the mid-support phase of the gait and loaded via the Achilles tendon over a broad range of functional loads (0 to 2943 N). Strains were recorded on the craniolateral, craniomedial, caudal, medial and lateral cortices at mid-diaphysis. Loading variations included the progressive elimination of the ligament and tendon along the caudal calcaneus. The results showed that the cranial cortex experiences longitudinal compressive strains that are nearly equal to the principal minimum strains and that the caudal cortex receives longitudinal tensile strains that are nearly equal to the principal maximum strains. With a 981 N load, the mean principal compressive strain on the cranial cortex was -636+/-344 micro(&egr;) (mean +/- s.d., N=9) and the mean principal tensile strain on the caudal cortex was 1112+/-68 micro;(&egr;)x (N=9). In contrast to the cranial and caudal cortices, principal strains in the medial and lateral cortices displayed relatively large deviations from the longitudinal axis (medial, 24 degrees cranial; lateral, 27 degrees caudal). Although shear strains predominated at all gauge sites, variations in maximum shear strains showed no apparent regional pattern or consistent regional predominance. The plantar ligament and tendon of the superficial digital flexor muscle were shown to have important load-sharing functions. These results demonstrate that the functionally loaded artiodactyl calcaneus generally behaves like a cantilevered beam with longitudinal compression and tension strains predominating in opposing cranial and caudal cortices, respectively. Differences in osteon remodeling rates, osteon morphology and mineral content reported previously between the cranial and caudal cortices correlate, in part, with the magnitudes of the principal compressive and tensile strains, respectively. However, material differences that distinguish the medial and lateral cortices from the cranial and caudal cortices could not be primarily attributed to locally increased shear strains as previously suggested. Variations in osteon and/or collagen fiber orientation may correlate more strongly with principal strain direction.

Age-related hypermineralization in the female proximal human femur.

Hip fracture incidence increases exponentially with age in virtually every human population that has been studied. In spite of this, relatively few studies have examined age-related changes in the metaphyseal cortex of the proximal femur. The present study investigates cortical aging changes in the female proximal femur, with particular reference to regions of hypermineralization. Thirty-three femora from Caucasian females were obtained at autopsy and analyzed using backscattered electron imaging. Variations in hypermineralized tissue area, cortical bone area, and porosity were quantified with standard stereological methods. Cortical width was quantified with digital calipers. Gender differences were examined by statistical comparison with previously published results. Hypermineralized tissue volume was significantly (P < 0.001) greater in elderly individuals. Hypermineralized tissue preferentially appeared near ligamentous or tendinous insertion sites, suggesting the hypermineralized tissue may be a calcified fibrocartilage. Cortical width significantly (P < 0.001) decreased with age and porosity significantly (P < 0.001) increased with age, however the changes were site-specific. The femoral neck and intertrochanteric cortices had a smaller change in cortical width and porosity with age than the diaphysis, but the femoral neck and intertrochanteric cortices had a larger increase in hypermineralized tissue. Comparison with previous data suggests that cortical aging in the proximal femur is similar between males and females and is unlikely to explain the higher incidence of fracture in females. However, the data strongly indicates that age-related changes in the femoral diaphysis cannot be directly extrapolated to either the femoral neck or intertrochanteric cortices.

Comparative micromotion of fully and proximally cemented femoral stems.

This investigation studied the differences of in vitro micromotion between two stem designs. The two stem types investigated were a proximally cemented stem with distal press fit and a fully cemented stem. After initial micromotion testing to 2250 N in simulated single leg stance and stair climb, six of each stem type were loaded dynamically for 1 million cycles at 950 N at 1 Hz. Micromotion studies were repeated. The two stem types had similar micromotion. For the single leg stance, fully cemented implant motion averaged (+/- 95% confidence) 18 +/- 8 microns toggle, 41 +/- 5 microns axial, and 59 +/- 22 microns rotation. Proximally cemented implant motion averaged 20 +/- 6 microns toggle, 42 +/- 6 microns axial, and 31 +/- 15 microns rotation. For the simulated stair climb, fully cemented implant motion averaged 24 +/- 10 microns toggle, 45 +/- 8 microns axial, and 92 +/- 32 microns rotation. Proximally cemented implant motion averaged 19 +/- 10 microns toggle, 42 +/- 9 microns axial, and 87 +/- 53 microns rotation. For both loading conditions, there were no significant differences measurable between the two systems. After dynamic testing of the fully cemented implants, there were no significant changes in the micromotion of either the toggle or the rotation, but an average of 18 microns increase of axial motion was measured in the fully cemented stem. For the proximally cemented implants, there were no significant changes after dynamic testing. This differences was not considered clinically significant because roentgen stereophotogrammetric analysis studies have shown that more than 4 mm of migration is required before clinical symptoms manifest. The protocol developed in this study may help provide a screening process to determine the stability of femoral stem designs before these devices are used clinically.

Influence of topography and specimen preparation on backscattered electron images of bone.

Backscattered electron (BSE) images of bone exhibit graylevel contrast between adjacent lamellae. Mathematical models suggest that interlamellar contrast in BSE images is an artifact due to topographic irregularities. However, little experimental evidence has been published to support these models, and it is not clear whether submicron topographical features will alter BSE graylevels. The goal of this study was to determine the effects of topography on BSE image mean graylevels and graylevel histogram widths using conventional specimen preparation techniques. White-light interferometry and quantitative BSE imaging were used to investigate the relationship between the BSE signal and specimen roughness. Backscattered electron image graylevel histogram widths correlated highly with surface roughness in rough preparations of homogeneous materials. The relationship between BSE histogram width and surface roughness was specimen dependent. Specimen topography coincided with the lamellar patterns within the bone tissue. Diamond micromilling reduced average surface roughness when compared with manual polishing techniques but did not significantly affect BSE graylevel histogram width. The study suggests that topography is a confounding factor in quantitative BSE analysis of bone. However, there is little quantitative difference between low-to-moderate magnification BSE images of bone specimens prepared by conventional polishing or diamond micromilling.

Errors in quantitative backscattered electron analysis of bone standardized by energy-dispersive x-ray spectrometry.

Backscattered electron (BSE) imaging has proven to be a useful method for analyzing the mineral distribution in microscopic regions of bone. However, an accepted method of standardization has not been developed, limiting the utility of BSE imaging for truly quantitative analysis. Previous work has suggested that BSE images can be standardized by energy-dispersive x-ray spectrometry (EDX). Unfortunately, EDX-standardized BSE images tend to underestimate the mineral content of bone when compared with traditional ash measurements. The goal of this study is to investigate the nature of the deficit between EDX-standardized BSE images and ash measurements. A series of analytical standards, ashed bone specimens, and unembedded bone specimens were investigated to determine the source of the deficit previously reported. The primary source of error was found to be inaccurate ZAF corrections to account for the organic phase of the bone matrix. Conductive coatings, methylmethacrylate embedding media, and minor elemental constituents in bone mineral introduced negligible errors. It is suggested that the errors would remain constant and an empirical correction could be used to account for the deficit. However, extensive preliminary testing of the analysis equipment is essential.