Intraskeletal variability in bone mass

For methodological or other reasons, a variety of skeletal elements are analyzed and subsequently used as a basis for describing general bone loss and mass. However, bone loss and mass may not be uniform within and among skeletal elements of the same individual because of biomechanical factors. We test the hypothesis that a homogeneity in bone mass exists among skeletal elements of the same individual. Measures indicative of bone mass were calculated from the midshafts of six skeletal elements from the same individuals (N = 41). The extent of intraskeletal variability in bone mass (relative cortical area) was then examined for the entire sample, according to age, sex, and pathological status. The results of the analysis showed that all measures reflect a heterogeneity in bone mass (P 相似文献   

Paleoepidemiological patterns of interpersonal aggression in a prehistoric central California population from CA‐ALA‐329

Interpersonal aggression is assessed paleoepidemiologically in a large skeletal population from the CA‐ALA‐329 site located on the southeastern side of San Francisco Bay, California. This comprehensive analysis included all currently recognized skeletal criteria, including craniofacial fracture, projectile injury, forearm fracture, and perimortem bone modification. Craniofacial injury is moderately common, showing an adult prevalence of 9.0% with facial lesions accounting for >50% of involvement. Clinical studies suggest that such separate evaluation of facial involvement provides a useful perspective for understanding patterns of interpersonal aggression. In this group male facial involvement is significantly greater than in females, paralleling the pattern found widely in contemporary populations as well as in African apes. When compared to other North American skeletal samples the prevalence of adult cranial vault injury (3.3%) and especially projectile injury (4.4%) are quite high. However, well documented populations from southern California show markedly higher prevalence for both types of skeletal markers of aggression. Forearm fracture is also assessed using a rigorous radiographic methodology and results suggest that these injuries are not reliable indicators of interpersonal aggression. Lastly, perimortem bone modification was not observed in this population, although it has been recorded from other (older) sites nearby. This study provides an evaluation of multiple skeletal markers of interpersonal aggression in the largest sample from a single site yet reported in North America and, joined with consideration of cultural context, helps further illuminate both geographic and temporal patterns of interpersonal aggression in California. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Bone strain magnitude is correlated with bone strain rate in tetrapods: implications for models of mechanotransduction

Hypotheses suggest that structural integrity of vertebrate bones is maintained by controlling bone strain magnitude via adaptive modelling in response to mechanical stimuli. Increased tissue-level strain magnitude and rate have both been identified as potent stimuli leading to increased bone formation. Mechanotransduction models hypothesize that osteocytes sense bone deformation by detecting fluid flow-induced drag in the bone''s lacunar–canalicular porosity. This model suggests that the osteocyte''s intracellular response depends on fluid-flow rate, a product of bone strain rate and gradient, but does not provide a mechanism for detection of strain magnitude. Such a mechanism is necessary for bone modelling to adapt to loads, because strain magnitude is an important determinant of skeletal fracture. Using strain gauge data from the limb bones of amphibians, reptiles, birds and mammals, we identified strong correlations between strain rate and magnitude across clades employing diverse locomotor styles and degrees of rhythmicity. The breadth of our sample suggests that this pattern is likely to be a common feature of tetrapod bone loading. Moreover, finding that bone strain magnitude is encoded in strain rate at the tissue level is consistent with the hypothesis that it might be encoded in fluid-flow rate at the cellular level, facilitating bone adaptation via mechanotransduction.     

Estimation of adult skeletal age‐at‐death using the Sugeno fuzzy integral

Age‐at‐death estimation of an individual skeleton is important to forensic and biological anthropologists for identification and demographic analysis, but it has been shown that the current aging methods are often unreliable because of skeletal variation and taphonomic factors. Multifactorial methods have been shown to produce better results when determining age‐at‐death than single indicator methods. However, multifactorial methods are difficult to apply to single or poorly preserved skeletons, and they rarely provide the investigator with information about the reliability of the estimate. The goal of this research is to examine the validity of the Sugeno fuzzy integral as a multifactorial method for modeling age‐at‐death of an individual skeleton. This approach is novel because it produces an informed decision of age‐at‐death utilizing multiple age indicators while also taking into consideration the accuracies of the methods and the condition of the bone being examined. Additionally, the Sugeno fuzzy integral does not require the use of a population and it qualitatively produces easily interpreted graphical results. Examples are presented applying three commonly used aging methods on a known‐age skeletal sample from the Terry Anatomical Collection. This method produces results that are more accurate and with smaller intervals than single indicator methods. Am J Phys Anthropol 2010. © 2009 Wiley‐Liss, Inc.     

Lactoferrin promotes bone growth

We have demonstrated bovine or human lactoferrin to be an anabolic factor in skeletal tissue. In vitro, lactoferrin stimulates the proliferation of bone forming cells, osteoblasts, and cartilage cells at physiological concentrations (above 0.1 microg/ml). The magnitude of this effect exceeds that observed in response to other skeletal growth factors such as IGF-1 and TGFbeta. DNA synthesis is also stimulated in a bone organ culture system likely reflecting the proliferation of cells of the osteoblast lineage. Lactoferrin is also a potent osteoblast survival factor. In TUNEL and DNA fragmentation assays, lactoferrin decreased apoptosis, induced by serum withdrawal, by up to 70%. In addition, lactoferrin has powerful effects on bone resorbing cells, osteoclasts, decreasing osteoclast development at concentrations > 1 microg/ml in a murine bone marrow culture system. However, lactoferrin did not alter bone resorption in calvarial organ culture, suggesting that it does not influence mature osteoclast function. In vivo, local injection of lactoferrin in adult mice resulted in increased calvarial bone growth, with significant increases in bone area and dynamic histomorphometric indices of bone formation after only 5 injections. Taken together, these data demonstrate that the naturally-occurring glycoprotein lactoferrin is anabolic to bone in vivo, an effect which is consequent upon its potent proliferative and anti-apoptotic actions in osteoblasts, and its ability to inhibit osteoclastogenesis. Lactoferrin may therefore have a physiological role in bone growth, and a potential therapeutic role in osteoporosis.     

Lateral angle: a method for sexing using the petrous bone

We report on the results of applying the so-called lateral angle method for sex determination on skeletal remains. The lateral angle denotes the angle of the internal auditory canal in relation to the medial surface of the petrous part of the temporal bone. The method involves making a small cast of the proximal part of the internal acoustic canal and determining the angle at which the canal opens up to the surface of the petrous bone. The method has the great advantage of utilizing one of the sturdiest bone elements of the human skeleton, and may thus be especially suited for analyses of very fragmented skeletal remains or cremated bones, where the petrous bone may still be readily recognizable. The method was tested using a forensic sample of 113 petrous bones with known sex. Intra- and interobserver testing was also performed. We found a statistically significant difference in angle size between males and females (mean angle size of males, 39.3 degrees ; mean angle size of females, 48.2 degrees ; P < 0.001). There was no bilateral difference in angle size. In blind trials, 83.2% of petrous bones were assigned to the correct sex. We also tested the lateral angle method against an archaeological skeletal sample. True sex was not known for this sample; instead, sexing had been carried out by assessing pelvic and cranial morphology in independent trials. We found a higher concordance between the lateral angle and "pelvic" sex than for lateral angle and "cranial" sex. Finally, we note that subadult sexing may also be possible with this method.     

Is amino acid racemization a useful tool for screening for ancient DNA in bone?

Many rare and valuable ancient specimens now carry the scars of ancient DNA research, as questions of population genetics and phylogeography require larger sample sets. This fuels the demand for reliable techniques to screen for DNA preservation prior to destructive sampling. Only one such technique has been widely adopted: the extent of aspartic acid racemization (AAR). The kinetics of AAR are believed to be similar to the rate of DNA depurination and therefore a good measure of the likelihood of DNA survival. Moreover, AAR analysis is only minimally destructive. We report the first comprehensive test of AAR using 91 bone and teeth samples from temperate and high-latitude sites that were analysed for DNA. While the AAR range of all specimens was low (0.02–0.17), no correlation was found between the extent of AAR and DNA amplification success. Additional heating experiments and surveys of the literature indicated that d/l Asx is low in bones until almost all the collagen is lost. This is because aspartic acid is retained in the bone within the constrained environment of the collagen triple helix, where it cannot racemize for steric reasons. Only if the helix denatures to soluble gelatin can Asx racemize readily, but this soluble gelatine is readily lost in most burial environments. We conclude that Asx d/l is not a useful screening technique for ancient DNA from bone.     

A novel purification method for multipotential skeletal stem cells

At least some cells within bone marrow stromal populations are multipotential (i.e., differentiate in vitro into osteoblasts, chondrocytes, and adipocytes) and thus designated skeletal stem cells (SSCs) or mesenchymal stem cells (MSCs) amongst other names. Recently, a subpopulation of stromal cells, notably osteoblasts or their progenitors, has been identified as a definitive regulatory component of the hematopoietic stem cell (HSC) niche. Thus, the development of methods for purifying not only SSCs but cells comprising the HSC niche is of interest. Here, we report a method for purifying a novel bone marrow‐derived population with a high frequency of osteoprogenitors and high expression levels of osteoblast differentiation markers (highly purified osteoprogenitors (HipOPs)) as well as markers of the bone niche for HSCs. In vivo transplantation experiments demonstrated that donor HipOPs differentiated into not only osteoblasts, osteocytes and cells around sinusoids but also hematopoietic cells. Thus, HipOPs represent a novel population for simultaneous reconstruction of bone and bone marrow microenvironments. J. Cell. Biochem. 108: 368–377, 2009. © 2009 Wiley‐Liss, Inc.     

Adult females and pubic bone growth

Previous research (Tague [1994] Am. J. Phys. Anthropol. 95:27–40) has shown an age effect in pubic bone length among adult women. Tague found that in three prehistoric Native American skeletal samples, women aged 18–24 had a significantly shorter linea terminalis than did women aged 25 and older. The purpose of this research is to determine whether such a difference can be discerned in other female skeletal samples. Three female skeletal samples were used in this analysis: 75 African-American and 42 European-American females aged 18–39 from the Hamann-Todd Collection (collected between 1893 and 1938; Iscan, 1990) and 99 African-American females aged 18–39 from the Terry Collection (collected between 1914 and 1965; Cobb, 1933; Iscan, 1990). Several chord measurements of pubic bone length along the linea terminalis were analyzed by one-tailed t-tests of the separate samples subdivided into two age groups: 18–24 and 25–39 years. Of 15 comparisons between age groups, none differed significantly by age group within each sample. It is concluded that the observed significant difference in pubic bone length in the Native American female skeletal samples cannot be replicated in other samples and that there is no age effect on pubic bone length in the samples tested in this analysis. Tague's findings reflect either the occurrence of late menarche in prehistoric populations or differential survivorship. Am J Phys Anthropol 106:323–328, 1998. © 1998 Wiley-Liss, Inc.     

Nondestructive sampling of human skeletal remains yields ancient nuclear and mitochondrial DNA

Museum curators and living communities are sometimes reluctant to permit ancient DNA (aDNA) studies of human skeletal remains because the extraction of aDNA usually requires the destruction of at least some skeletal material. Whether these views stem from a desire to conserve precious materials or an objection to destroying ancestral remains, they limit the potential of aDNA research. To help address concerns about destructive analysis and to minimize damage to valuable specimens, we describe a nondestructive method for extracting DNA from ancient human remains. This method can be used with both teeth and bone, but it preserves the structural integrity of teeth much more effectively than that of bone. Using this method, we demonstrate that it is possible to extract both mitochondrial and nuclear DNA from human remains dating between 300 BC and 1600 AD. Importantly, the method does not expose the remains to hazardous chemicals, allowing them to be safely returned to curators, custodians, and/or owners of the samples. We successfully amplified mitochondrial DNA from 90% of the individuals tested, and we were able to analyze 1-9 nuclear loci in 70% of individuals. We also show that repeated nondestructive extractions from the same tooth can yield amplifiable mitochondrial and nuclear DNA. The high success rate of this method and its ability to yield DNA from samples spanning a wide geographic and temporal range without destroying the structural integrity of the sampled material may make possible the genetic study of skeletal collections that are not available for destructive analysis.     

Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.     

Label‐free quantitative proteome analysis of skeletal tissues under mechanical load

Skeletal tissue has the capability to adapt its mass and structure in response to mechanical stress. However, the molecular mechanism of bone and cartilage to respond to mechanical stress are not fully understood. A label‐free quantitative proteome approach was used for the first time to obtain a global perspective of the response of skeletal tissue to mechanical stress. Label‐free quantitative analysis of 1D‐PAGE‐LC/MS/MS based proteomics was applied to identify differentially expressed proteins. Differential expression analysis in the experimental groups and control group showed significant changes for 248 proteins including proteins related to proliferation, differentiation, regulation of signal transduction and energy metabolic pathways. Fluorescence labeling by incorporation of alizarin/calcein in newly formed bone minerals qualitatively demonstrated new bone formation. Skeletal tissues under mechanical load evoked marked new bone formation in comparison with the control group. Bone material apposition was evident. Our data suggest that 39 proteins were assigned a role in anabolic process. Comparisons of anabolic versus catabolic features of the proteomes show that 42 proteins were related to catabolic. In addition, some proteins were related to regulation of signal transduction and energy pathways, such as tropomyosin 4, fibronectin 1, and laminin, might be new molecular targets that are responsive to mechanical force. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the effects of mechanical force on skeletal tissue. J. Cell. Biochem. 108: 600–611, 2009. © 2009 Wiley‐Liss, Inc.     

Differential skeletal preservation at Windover Pond: causes and consequences

In this paper, we evaluate the causes of differential skeletal preservation in the Windover Pond skeletal series (8BR246). We collected data on sex and age for approximately 110 individuals, and calculated a preservation score for each individual based on the presence of 80 skeletal landmarks. Our research questions evaluated the relationship between bone preservation and individual age and sex, and between the presence of preserved brain material and skeletal preservation, and the effects of burial location on bone preservation. The results indicate variability in average preservation for the sample (micro = 0.53, SD = 0.22) with an apparent lack of sex-specific (P = 0.79) or age-specific (P = 0.37) differences in preservation. The relationship between brain and skeletal preservation (P = 0.15) was not significant. The horizontal distribution of burials was not significantly correlated with skeletal preservation (north: r = -0.10, P = 0.93; east: r = 0.09, P = 0.45); however, vertical depth was a significant predictor of preservation (r = -0.31, P = 0.005), indicating that skeletal preservation decreased as burials were located closer to the ground surface. The observed variability in preservation scores may be related to the partial drying and resubmergence of the uppermost burials for the last few millennia. Comparison of Windover element-specific survival rates with previous analyses based on terrestrial samples (Galloway et al. [1997] Forensic taphonomy, Boca Raton: CRC Press; Waldron [1987] Death, decay and reconstruction, Manchester: Manchester University Press; Willey et al. [1997] Am J Phys Anthropol 104:513-528) affirms the relationship between element weight or density and bone survival. The unique taphonomic context of our study sample effected little change in bone deterioration processes.     

Measuring and interpreting age‐related loss of vertebral bone mineral density in a medieval population

This study investigates the age‐ and sex‐related patterns in vertebral bone mineral density (BMD) and the relationship between BMD and vertebral osteophytosis (VO), using a specialized peripheral densitometer in a skeletal sample excavated from the British medieval village Wharram Percy. A total of 58 individuals were divided by sex into three broad age categories (18–29, 30–49, 50+ years.). Each fourth intact vertebral centra was scored for VO and 5‐mm thick coronal sections scanned in a specialized peripheral densitometer (GE Lunar Piximus DXA). Changes in BMD associated with age, sex, and VO severity were examined in the whole vertebral section, a strictly trabecular region, and a primarily cortical region of bone separately. Significant change in vertebral BMD was found to occur by middle age with little or no statistical change in BMD between middle and old age. Females appear to suffer greater bone loss at an earlier age with no change in BMD between middle and old age, whereas males show a more steady loss of BMD across the age groups. The bone mineral content and BMD of the cortical region is higher in individuals with pronounced/severe osteophytosis. The unusual age‐ and sex‐related patterns of change in vertebral BMD at Wharram Percy are compared with the patterns of age‐related change from recent longitudinal population‐based studies. The results emphasize the different pattern of bone loss in young adulthood seen in trabecular regions of the skeleton and highlight the importance of consideration of degenerative joint disease in BMD studies. The influence of lifestyle factors on vertebral BMD in this medieval population is also discussed. Am J Phys Anthropol 2009. © 2009 Wiley‐Liss, Inc.     

Body size and joint posture in primates

Body mass has been shown in experimental and comparative morphological studies to have a significant effect on joint posture in major limb joints. The generalizability of experimental studies is limited by their use of small sample sizes and limited size ranges. In contrast, while comparative morphological studies often have increased sample sizes, the connection between joint posture and morphological variables is often indirect. The current study infers joint postures for a large sample of primates using an experimentally validated method, and tests whether larger primates use more extended joint postures than smaller species. Postures are inferred through the analysis of patterns of subchondral bone apparent density on the medial femoral condyle. Femora from 94 adult wild‐shot individuals of 28 species were included. Apparent density measurements were obtained from CT scans using AMIRA software, and the angular position of the anterior‐most extent of the region of maximum apparent density on the medial femoral condyle was recorded. In general, the hypothesis that larger‐bodied primates use more extended knee posture was supported, but it should be noted that considerable variation exists, particularly at small body sizes. This indicates that smaller species are less constrained by their body size, and their patterns of apparent density are consistent with a wide range of knee postures. The size‐related increase in inferred joint posture was observed in most major groups of primates, and this observation attests to the generalizability of Biewener's model that relates body size and joint posture. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of parity on bone mineral density in female rhesus macaques from Cayo Santiago

This cross-sectional study investigates the relationship between parity, bone mineral density, and spontaneous osteopenia/osteoporosis in a large skeletal population of female rhesus macaques (Macaca mulatta) from the free-ranging colony of Cayo Santiago, Puerto Rico. The sample consists of 119 mature female monkeys aged 4.0-22.2 years at time of death. The data consist of measurements of bone mineral content (BMC) and bone mineral density (BMD), obtained from dual-energy X-ray absorptiometry (DEXA) of the last lumbar vertebra. After controlling for age, there is a significant increase in BMD of the spine with increasing parity (P = 0.0006), up to a parity of 7 offspring. Thus, high parity initially has a positive effect on BMD in female rhesus monkeys, but this positive effect disappears with parities that are greater than 7 offspring. After controlling for parity, however, age has a negative (P = 0.015) effect on BMD, beginning several years after the attainment of peak BMD (age 9.5 years). Thus, it appears that parity initially mitigates the effects of aging, but the positive effect of parity on BMD is eventually overwhelmed by the aging process. Mean BMC and BMD values are higher in parous females compared to nulliparous females in the same age range. Similarly, females with low parity have significantly lower mean BMD values than do age-matched high-parity controls, and the frequency of osteopenia and osteoporosis is greater in low-parity females. Forty-three percent (43%) of the osteopenic/osteoporotic females in the sample are members of the low-parity group, even though it composes only 13% (16/119) of the entire sample. This study demonstrates that the free-ranging female rhesus monkeys from Cayo Santiago are a good nonhuman primate model for the study of bone mineral density, parity, osteopenia, and osteoporosis.     

Hibernation does not reduce cortical bone density,area or second moments of inertia in woodchucks (Marmota monax)

Long periods of inactivity in most mammals result in bone loss that may not be completely recoverable during an individual's lifetime regardless of future activity. Prolonged inactivity is normal during hibernation, but it remains uncertain whether hibernating mammals suffer decreased bone properties after hibernation that affects survival. We test the hypothesis that relative cortical area (C_A), apparent density, bone area fraction (B.Ar/T.Ar), and moments of inertia do not differ between museum samples of woodchucks (Marmota monax) collected before and after hibernation. We used peripheral quantitative computed tomography to examine bone geometry in the femur, tibia, humerus and mandible. We see little evidence for changes in bone measures with hibernation supporting our hypothesis. In fact, when including subadults to increase sample sizes and controlling age statistically, we observed a trend toward increased bone properties following hibernation. Diaphyses were significantly denser in the humerus, femur, and tibia after hibernation, and relative mandibular cortical area was significantly larger. Similarly, relative mechanical indices were significantly larger in the mandible after hibernation. Although tests of individual measures in many cases were not significantly different prehibernation versus posthibernation, the overall pattern of average increase posthibernation was significant for relative C_A and densities as well as relative diaphyseal mechanical indices when examining outcomes collectively. The exception to this pattern was a reduction in metaphyseal trabecular bone following hibernation. Individually, only humeral B.Ar/T.Ar was significantly reduced, but the average reduction in trabecular measures post‐hibernation was significant when examined collectively. Because the sample included subadults, we suggest that much of the increased bone relates to their continued growth during hibernation. Our results indicate that woodchucks are more similar to large hibernators that maintain skeletal integrity compared to smaller‐bodied hibernators that may lose bone. This result suggests a potential size‐related trend in bone response to hibernation across mammals. J. Morphol., 2012. © 2012Wiley Periodicals, Inc.     

Ancient DNA studies

Now that the hype surrounding Jurassic Park has settled down and we have become relatively used to dramatic headlines announcing the recovery of DNA from exotic fossilized remains, scientists working on ancient DNA are beginning to reflect on the long-term prospects and implications of the subject.¹ The science of ancient DNA has grown exponentially since its birth only ten years ago, and despite serious technical difficulties, it promises to become a revolutionary research tool in anthropology and molecular evolution. The use of bone DNA typing in particular has already yielded useful insights into Polynesian prehistory as well as spectacular applications in the forensic identification of skeletal remains.     

人骨形成蛋白3在昆虫杆状病毒表达系统中的表达、纯化及其诱骨活性检测

为了在昆虫杆状病毒系统中表达人骨形成蛋白 3(humanbonemorphogeneticproteoin3,hBMP3) ,检测表达产物的诱骨活性 .将hBMP3全长cDNA 1416bp克隆入转移载体K8中 ,再与病毒DNA经脂质体包裹后转染昆虫细胞Sf9.重组病毒经蓝白筛选后 ,用PCR方法扩增目的基因片段进行初步鉴定 .收集重组病毒的转染上清 ,肝素亲和层析纯化目的蛋白 .SDS PAGE和Western印迹进一步鉴定此蛋白 .体外培养MC3T3 E1细胞 ,经目的蛋白刺激后 ,检测细胞内碱性磷酸酶 (ALP)活性 .并将目的蛋白进行小鼠体内肌肉包埋实验 ,检测其异位诱骨活性 .结果显示 ,肝素亲和层析可以收集 1个高的洗脱峰 .SDS PAGE显示 ,非还原型样本为 32kD的二聚体蛋白带和少量 16kD单体蛋白带 ;还原型样本仅有相应大小的单体蛋白带 ,纯度达 80 % .Western印迹在相应位置呈阳性染色 .此蛋白刺激小鼠成纤维细胞 4 8h后 ,胞内ALP的活性增高 ,经rhBMP3作用后的细胞MTT染色减弱 .在小鼠股部肌肉内包埋蛋白样品 2～ 3周 ,组织学检测有成骨组织生成 .说明hBMP3在此套系统中获得了表达 .表达产物在体外可以刺激成骨细胞的分化 ,却抑制细胞的增殖 .小鼠体内异位诱骨实验进一步证实表达产物具有成骨活性     

A Three-Dimensional Histological Method for Direct Determination of the Number of Trabecular Termini in Cancellous Bone

Osteoporotic fractures occur frequently in aging populations. Established methods for analyzing microarchitecture indicate that cancellous bone loss in the elderly is associated with progressive reduction in the connectivity of the trabecular network. This disconnection may explain the increased skeletal fragility that is sometimes out of proportion to the amount of bone lost. Connectivity, however, is difficult to measure and usually requires indirect methods. We describe development of a simple, inexpensive and direct procedure for counting sites of trabecular disconnection. The method is based upon preparation of 300-500 fjim thick slices of methylmethacrylate embedded material rather than the more usual thin 8 μm. histological sections. The marrow tissue is retained within the thick slice; this is essential for conservation of any detached bone fragments. In such preparations differential superficial staining of the upper and lower surfaces with alizarin red and light green, respectively, allows the two-dimensional image to be viewed at the same time as its three-dimensional counterpart. In this way, “real” (i. e., unstained) trabecular termini can be distinguished from “apparent” (i. e., stained red or green) termini that are artifacts of the plane of section. Partly polarized light enhances the microscope image. The method does not destroy the material for subsequent bone histomorphom-etry and, therefore, may be a useful adjunct to iliac bone biopsy analysis in studies of metabolic bone disease.