The skeletal attachment of tendons--tendon "entheses"

Tendon entheses can be classed as fibrous or fibrocartilaginous according to the tissue present at the skeletal attachment site. The former can be "bony" or "periosteal", depending on whether the tendon is directly attached to bone or indirectly to it via the periosteum. At fibrocartilaginous entheses, the uncalcified fibrocartilage dissipates collagen fibre bending and tendon narrowing away from the tidemark; calcified fibrocartilage anchors the tendon to the bone and creates a diffusion barrier between the two. Where there are additional fibrocartilaginous specialisations in the tendon and/or bone next to the enthesis, an "enthesis organ" is created that reduces wear and tear. Little attention has been paid to bone at entheses, despite the obvious bearing this has on the mechanical properties of the interface and the clinical importance of avulsion fractures. Disorders at entheses (enthesopathies) are common and occur in conditions such as diffuse idiopathic skeletal hyperostosis and the seronegative spondyloarthropathies. They are also commonly seen as sporting injuries such as tennis elbow and jumper's knee.     

Enthesopathies--proposal of a standardized scoring method and applications

Enthesopathies are alterations that could be present at entheses. Two types of enthesopathies have been defined: osteophytic (OF) and osteolytic (OL). In the present paper, we propose a standardized method to score the degree of development of each form of enthesopathy. With this method, the intra- and interobserver errors are less than 50%. The standard was used to study a sample (113 individuals) deriving from osteological collections from the late XIX-early XX century. Information about the age, sex and occupation of the individuals is available. This study demonstrated an effect of age on the form and the degree of development of enthesopathies. The influence of factors related to sex and occupation cannot be excluded. Therefore, functional interpretations of data on enthesopathies in osteoarchaeological series must take account of the estimated age and sex of the specimens and the distribution of the lesions within a single skeleton.     

Bone microarchitecture at muscle attachment sites: The relationship between macroscopic scores of entheses and their cortical and trabecular microstructural design

The studies of entheses in bioarchaeology attempted to reconstruct the habitual physical activities of past populations. However, the studies of microarchitecture of the underlying bone are still lacking despite well‐known potential of bone internal microarchitecture to reflect mechanical loading. It is unknown whether different morphological expressions of entheseal changes (ECs) correlate with the microstructural characteristics of the underlining bone. This study analyzed bone microstructural characteristics at the entheses. Our focus was on examining the possible successive nature of the three‐stage scale of entheseal macroscopic changes by comparing EC scores with the microarchitectural features at the attachment sites. The study was based on the hypothesis that mechanical loading influences the microarchitecture of the bone at the attachment site. The bone samples were taken from 24 adult male skeletons from medieval cemeteries in Serbia, with different macroscopic expression score of EC. We evaluated the macroscopic and microscopic appearance of four entheses of the lower limbs (origin of the soleus muscle and the insertions of the adductor magnus, gluteus maximus, and iliopsoas muscles). The specimens were scanned using microcomputed tomography (Scanco µCT 40). Our data showed a lack of consistent correlation between stages of the macroscopic scoring systems with microarchitecture at the entheses, only cortical thickness was significantly different between EC stages. Analyzing relationship between trabecular and cortical bone microstructure we found correlations between cortical and trabecular variables only in Stage C. Results of our study suggest that macroscopic EC might not represent distinct successive phases in bone adaptation to mechanical loading. Am J Phys Anthropol 157:81–93, 2015. © 2014 Wiley Periodicals, Inc.     

Contribution of PTHrP to mechanical strain-induced fibrochondrogenic differentiation in entheses of Achilles tendon of miniature pigs

Background

Fibrochondrocytes are involved in entheses repair, but their response to mechanical strain (MS) is ill known.

Objective

To determine if parathyroid hormone-related protein (PTHrP) expression in fibrochondrocytes from fibrocartilaginous entheses is modulated by MS, and to further observe the regulatory effects of human (h) PTHrP on fibrochondrocyte differentiation.

Methods

Fibrochondrocytes from fibrocartilaginous entheses of Guizhou miniature pig?s Achilles tendon were submitted or not to MS (4%, 8% or 12% cyclic tensile strain; 1 Hz). Fibrochondrocytes were also exposed to: cyclopamine (Indian hedgehog (Ihh) inhibitor) (10 μM), hPTHrP (10 nM) or cyclopamine/hPTHrP (cyclopamine 10uM+hPTHrP 10 nM). Types I, II and X collagen and PTHrP expressions were measured by real-time RT-PCR and Western blot.

Result

Under 4% strain load for 12 h, types I and II collagen mRNA expressions were increased (+324% and +659%, P<0.001), while type X collagen was decreased (−89%, P<0.001). At 12%, types I and II collagen mRNA expressions were decreased (−62% and −62%, P<0.001), while type X collagen was increased (+375%, P<0.05). Under 4% strain load, PTHrP mRNA expression was increased in relation with strain duration (from 3 to 12 h: +168%, P<0.001), while at 12%, PTHrP expression decreased with time (from 3 to 12 h: −81%, P<0.001). Using cyclopamine for 24 h, PTHrP mRNA expression was significantly decreased (−88%, P<0.05), types I and II collagen were decreased (−90% and −82%, P<0.001), and type X collagen was increased (+261%, P<0.001).

Conclusions

Dynamic MS modulate PTHrP expressions. Thus, PTHrP might play an important role in fibrochondrocyte differentiation, indirectly revealing a role in entheses? formation and repair.     

The effect of age, sex, and physical activity on entheseal morphology in a contemporary Italian skeletal collection

Entheseal changes are traditionally included in a large array of skeletal features commonly referred to as "skeletal markers of activity." However, medical studies and recent anthropological analyses of identified skeletal series suggest a complex combination of physiological and biomechanical factors underlying the variability of such "markers." The aim of this study is to examine the relationship between age, sex, physical activity, and entheseal variability. To this end, 23 postcranial entheses are examined in a large (N = 484) Italian contemporary skeletal series using standardized scoring methods. The sample comprises subjects of known age, sex and, mostly, occupation. Results show a strong relationship between age and entheseal changes. Differences between sexes are also highlighted, while the effects of physical activity appear moderate. Altogether, our study indicates that entheseal morphology primarily reflects the age of an individual, while correlation with lifetime activity remains ambiguous.     

Nano-hydroxyapatite-thermally denatured small intestine sub-mucosa composites for entheses applications

The objective of the present in vitro study was to estimate the adhesion strength of nanometer crystalline hydroxyapatite (HA)-small intestine sub-mucosa (SIS) composites on model implant surfaces. Techniques of thermal denaturation (60 degrees C, 20 min) of SIS were used to enhance the adhesion strength of entheses materials to underlying implants. Specifically, results indicated that the adhesion strength of thermally denatured SIS was 2-3 times higher than that for normal unheated SIS. In addition, aqua-sonicated, hydrothermally treated nano-HA dispersions enhanced the adhesion strength of SIS on implant surfaces. Importantly, results of the present study demonstrated that human skeletal muscle cell (hSkMC) numbers were not affected by thermally denaturing SIS in nano-HA composite coatings; however, they increased on aqua-sonicated nano-HA/SIS composites compared with SIS alone. Interestingly, thermally denatured SIS that contained aqua-sonicated, hydrothermally treated nano-HA decreased human osteoblasts (hOBs) numbers compared with respective unheated composites; all other composites when thermally denatured did not influence hOB numbers. Results also showed that the number of hOBs increased on nano-HA/SIS composites compared with SIS composites alone. Human mesenchymal stem cell (hMSC) numbers were not affected by the presence of nano-HA in SIS composites. For these reasons, the collective results of this in vitro study demonstrated a technique to increase the coating strength of entheses coatings on implant surfaces (using thermally denatured SIS and aqua-sonicated, hydrothermally prepared nano-HA) while, at the same time, supporting cell functions important for entheses regeneration.     

Mobility and subsistence economy: a diachronic comparison between two groups settled in the same geographical area (Liguria, Italy)

The purpose of this article is to investigate temporal shifts in skeletal robusticity to infer behavioral changes in two populations (Neolithic, NEOL and Medieval, MED) settled in the same geographic area but involved in different subsistence economies (pastoralism and coastal resources exploitation). This comparison allows us to test the hypothesis that occupational stress and mobility in the same environment produce predictable changes in the robusticity of both upper and lower limbs. Results show a lower degree of humeral robusticity and a similar degree of humeral asymmetry in the two sexes in the MED population. These results are consistent with the relatively less stressful subsistence economy documented in the MED population relative to that of the NEOL. Lower limb results suggest that femoral robusticity does not correlate directly with the level of logistical mobility, but is instead due to the summation of several diverse factors that place biomechanical loads on the hindlimb, particularly unevenness of the terrain. However, female femoral gracility seems to indicate that below a certain "threshold" of mobility, i.e., movement over the natural terrain, terrain conformation is no longer the main contributing factor to femoral robusticity. The femoral shape index I(x)/I(y) declines through time, particularly in males. This agrees with the expected mobility of the samples based on archaeological and historical data, providing further evidence on the reliability of this index in inferring terrestrial mobility.     

3D Microstructural Architecture of Muscle Attachments in Extant and Fossil Vertebrates Revealed by Synchrotron Microtomography

Background

Firm attachments binding muscles to skeleton are crucial mechanical components of the vertebrate body. These attachments (entheses) are complex three-dimensional structures, containing distinctive arrangements of cells and fibre systems embedded in the bone, which can be modified during ontogeny. Until recently it has only been possible to obtain 2D surface and thin section images of entheses, leaving their 3D histology largely unstudied except by extrapolation from 2D data. Entheses are frequently preserved in fossil bones, but sectioning is inappropriate for rare or unique fossil material.

Methodology/Principal Findings

Here we present the first non-destructive 3D investigation, by propagation phase contrast synchrotron microtomography (PPC-SRµCT), of enthesis histology in extant and fossil vertebrates. We are able to identify entheses in the humerus of the salamander Desmognathus from the organization of bone-cell lacunae and extrinsic fibres. Statistical analysis of the lacunae differentiates types of attachments, and the orientation of the fibres, reflect the approximate alignment of the muscle. Similar histological structures, including ontogenetically related pattern changes, are perfectly preserved in two 380 million year old fossil vertebrates, the placoderm Compagopiscis croucheri and the sarcopterygian fish Eusthenopteron foordi.

Conclusions/Significance

We are able to determine the position of entheses in fossil vertebrates, the approximate orientation of the attached muscles, and aspects of their ontogenetic histories, from PPC-SRµCT data. Sub-micron microtomography thus provides a powerful tool for studying the structure, development, evolution and palaeobiology of muscle attachments.     

The Influence of Life History and Sexual Dimorphism on Entheseal Changes in Modern Humans and African Great Apes

Entheseal changes have been widely studied with regard to their correlation to biomechanical stress and their usefulness for biocultural reconstructions. However, anthropological and medical studies have demonstrated the marked influence of both age and sex on the development of these features. Studies of entheseal changes are mostly aimed in testing functional hypotheses and are mostly focused on modern humans, with few data available for non-human primates. The lack of comparative studies on the effect of age and sex on entheseal changes represent a gap in our understanding of the evolutionary basis of both development and degeneration of the human musculoskeletal system. The aim of the present work is to compare age trajectories and patterns of sexual dimorphism in entheseal changes between modern humans and African great apes. To this end we analyzed 23 postcranial entheses in a human contemporary identified skeletal collection (N = 484) and compared the results with those obtained from the analysis of Pan (N = 50) and Gorilla (N = 47) skeletal specimens. Results highlight taxon-specific age trajectories possibly linked to differences in life history schedules and phyletic relationships. Robusticity trajectories separate Pan and modern humans from Gorilla, whereas enthesopathic patterns are unique in modern humans and possibly linked to their extended potential lifespan. Comparisons between sexes evidence a decreasing dimorphism in robusticity from Gorilla, to modern humans to Pan, which is likely linked to the role played by size, lifespan and physical activity on robusticity development. The present study confirms previous hypotheses on the possible relevance of EC in the study of life history, pointing moreover to their usefulness in evolutionary studies.     

Metatarsal robusticity in primates and a few other plantigrade mammals

By using a new technique for determining relative metatarsal robusticity, the distribution of the 1+5 pattern (in which M5 is the second most robust metatarsal after M1) and 15 pattern (in which M5 is the least robust of all metatarsals) was established in primates and a few other plantigrade mammals. The first pattern is associated with a terrestrial and the second with an arboreal substrate. Robusticity formulae are not connected with specific locomotor patterns, but a total robusticity quotient is associated with these patterns and substrate preference as well. Changes in substrate preference are accompanied by changes of total robusticity, an increased number of permutations and ultimately a change of the robusticity pattern. Intermetatarsal robusticity gradients are related to the direction and intensity of muscular activity. A combined analysis of all factors can reveal a great deal of the locomotor history of a taxon.Also ofThe Institute of Applied Biology, New York.     

Enthesis bilateral asymmetry in humans and African apes

Entheses (skeletal muscle and tendon attachment sites) have often been used to infer handedness and activity variability among human populations. However, the specific roles that intensity vs. frequency of muscle contractions play in modifying entheses are not well understood and the assumption that entheses reflect muscle activity levels has been challenged. This study explores the effect of habitual muscular activity on enthesis morphology in humans and African apes by investigating bilateral asymmetry in the forelimbs and hindlimbs of these taxa. Humans have generally more developed entheses in the lower limb while African apes have generally more developed entheses in the forelimbs. All species studied have more asymmetric forelimbs than hindlimbs except humans that show more asymmetrical expression of bony spurs in the lower limbs than in the upper limbs. When comparing species, humans are always more asymmetric in ethesis development than apes for both the forelimbs and hindlimbs, which reflects the relatively greater asymmetry in limb use in humans and the more symmetric use in apes. Enthesis development may reflect cross-symmetry patterns in humans and, more subtly, a moderate handedness in apes during manipulative activities. This study suggests that enthesis morphology provides information on muscle activity levels, with greater development of entheses associated with more habitual or powerful muscle use. The general similarity of ape and human responses to muscle activity suggests that muscle activity influenced enthesis development in Plio-Pleistocene hominins and that interpretation of muscle markings in these fossils can provide data for functional inferences in these extinct species.     

Metatarsal robusticity in bipedal rats

All metatarsals have a significantly greater robusticity in the male than in the female rat. The robusticity formula of the rat's foot is 1 > 5 > 2 > 3 > 4. In bipedal rats that formula remains unchanged, but the robusticity of the metatarsals is increased especially in females. The tripod arrangement of the human foot with its particular robustness of the marginal metatarsals 1 and 5 and a strong calcaneum has been related to upright posture. The similar robusticity pattern in the rat's marginal metatarsals 1 and 5 raises the question of whether that part of the formula might not represent a more general plantigrade pattern.     

3D representation and analysis of enthesis morphology

This comparison of methods for assessing the development of muscle insertion sites, or entheses, suggests that three‐dimensional (3D) quantification of enthesis morphology can produce a picture of habitual muscle use patterns in a past population that is similar to one produced by ordinal scores for describing enthesis morphology. Upper limb skeletal elements (humeri, radii, and ulnae) from a sample of 24 middle‐aged adult males from the Pottery Mound site in New Mexico were analyzed for both fibrous and fibrocartilaginous enthesis development with three different methods: ordinal scores, two‐dimensional (2D) area measurements, and 3D surface areas. The methods were compared using tests for asymmetry and correlations among variables in each quantitative data set. 2D representations of enthesis area did not agree as closely as ordinal scores and 3D surface areas did regarding which entheses were significantly asymmetrical. There was significant correlation between 3D and 2D data, but correlation coefficients were not consistently high. Intraobserver error was also assessed for the 3D method. Cronbach's alpha values fell between 0.68 and 0.73, and error rates for all entheses fell between 10% and 15%. Marginally acceptable intraobserver error and the analytic versatility of 3D images encourage further investigation of using 3D scanning technology for quantifying enthesis development. Am J Phys Anthropol 152:417–424, 2013. © 2013 Wiley Periodicals, Inc.     

Requirements for Hedgehog, a Segmental Polarity Gene, in Patterning Larval and Adult Cuticle of Drosophila

Mutations of the hedgehod gene are generally embryonic lethal, resulting in a lawn of denticles on the ventral surface. In strong alleles, no segmentation is obvious and the anteroposterior polarity of ventral denticles is lost. Temperature shift analysis of a temperature-sensitive allele indicates an embryonic activity period for hedgehod between 2.5 and 6 hr of embryonic development (at 25 degrees) and a larval/pupal period from 4 to 7 days of development (at 25 degrees). Mosaic analysis of hedgehod mutations in the adult cuticle indicates a series of defined defects associated with the failure of appropriate hedgehod expression. In particular, defects in the distal portions of the legs and antenna occur in association with homozygous hedgehog clones in the posterior compartment of those structures. Because the defects are associated with homozygous clones, but are not co-extensive, a type of "domineering" nonautonomy is proposed for the activity of the hedgehog gene.     

Exploring the relationship between entheseal changes and physical activity: A multivariate study

Analyses of entheseal changes (EC) in identified skeletal samples employ a common research strategy based on the comparison between occupations grouped on the basis of shared biomechanical and/or social characteristics. Results from this approach are often ambiguous, with some studies that point to differences in EC between occupational samples and others failing to provide evidence of behavioral effects on EC. Here we investigate patterns of EC among documented occupations by means of a multivariate analysis of robusticity scores in nine postcranial entheses from a large (N = 372) contemporary skeletal sample including specimens from one Italian and two Portuguese identified collections. Data on entheseal robusticity, analyzed by pooled sides as well by separated sides and levels of asymmetry, are converted in binary scores and then analyzed through nonlinear principal component analysis and hierarchical cluster analysis. Results of these analyses are then used for the classification of occupations. Differences between occupational classes are tested by MANOVA and pairwise Hotelling's test. Results evidence three classes which separate occupations related to farming, physically demanding but generalized occupation, and physically undemanding occupations, with the more consistent differences between the first and the last classes. Our results are consistent with differences in biomechanical behavior between the occupations included in each class, and point to the physical and social specificity of farming activities. On the other hand, our study exemplifies the usefulness of alternative analytical protocols for the investigation of EC, and the value of research designs devoid of a priori assumptions for the test of biocultural hypotheses. Am J Phys Anthropol 156:215–223, 2015. © 2014 Wiley Periodicals, Inc.     

Enthesopathies as occupational stress markers: Evidence from the upper limb

Enthesopathies—that is, “musculo‐skeletal stress markers”—are frequently used to reconstruct past lifestyles and activity patterns. Relatively little attention has been paid in physical anthropology to methodological gaps implicit in this approach: almost all methods previously employed neglect current medical insights into enthesopathies and the distinction between healthy and pathological aspects has been arbitrary. This study presents a new visual method of studying fibrocartilaginous enthesopathies of the upper limb (modified from Villotte: Bull Mém Soc Anthropol Paris n.s. 18 (2006) 65–85), and application of this method to 367 males who died between the 18th and 20th centuries, from four European identified skeletal collections: the Christ Church Spitalfields Collection, the identified skeletal collection of the anthropological museum of the University of Coimbra, and the Sassari and Bologna collections of the museum of Anthropology, University of Bologna. The analysis, using generalized estimating equations to model repeated binary outcome variables, has established a strong link between enthesopathies and physical activity: men with occupations involving heavy manual tasks have significantly (P‐value < 0.001) more lesions of the upper limbs than nonmanual and light manual workers. Probability of the presence of an enthesopathy also increases with age and is higher for the right side compared with the left. Our study failed to distinguish significant differences between the collections when adjusted for the other effects. It appears that enthesopathies can be used to reconstruct past lifestyles of populations if physical anthropologists: 1) pay attention to the choice of entheses in their studies and 2) use appropriate methods. Am J Phys Anthropol, 2010. © 2009 Wiley‐Liss, Inc.     

Biomechanics of cross-sectional size and shape in the hominoid mandibular corpus

Mandibular cross sections of Pan, Pongo, Gorilla, Homo, and two fossil specimens of Paranthropus were examined by computed tomography (CT) to determine the biomechanical properties of the hominoid mandibular corpus. Images obtained by CT reveal that while the fossil hominids do not differ significantly from extant hominoids in the relative contribution of compact bone to total subperiosteal area, the shape of the Paranthropus corpora indicates that the mechanical design of the robust australopithecine mandible is fundamentally distinct from that of modern hominoids in terms of its ability to resist transverse bending and torsion. It is also apparent that, among the modern hominoids, interspecific and sexual differences in corpus shape are not significant from a biomechanical perspective. While ellipse models have been used previously to describe the size, shape, and subsequent biomechanical properties of the corpus, the present study shows that such models do not predict the biomechanical properties of corpus cross-sectional geometry in an accurate or reliable manner. The traditional "robusticity" index of the mandibular corpus is of limited utility for biomechanical interpretations. The relationship of compact bone distribution in the corpus to dimensions such as mandibular length and arch width may provide a more functionally meaningful definition of mandibular robusticity.     

Which measures of diaphyseal robusticity are robust? A comparison of external methods of quantifying the strength of long bone diaphyses to cross‐sectional geometric properties

Measures of diaphyseal robusticity have commonly been used to investigate differences in bone strength related to body size, behavior, climate, and other factors. The most common methods of quantifying robusticity involve external diameters, or cross-sectional geometry. The data derived from these different methods are often used to address similar research questions, yet the compatibility of the resulting data has not been thoroughly tested. This study provides the first systematic comparison of externally derived measures of postcranial robusticity, with those based upon cross-sectional geometry. It includes sections taken throughout the skeleton, comparisons of prediction errors associated with different measurements, and analysis of the implications of different methods of body size standardization on the prediction of relative bone strength. While the results show reasonable correlations between diaphyseal diameters and strengths derived from cross-sectional geometry, considerable prediction errors are found in many cases. A new approach to externally based quantification of diaphyseal robusticity based upon moulding of sub-periosteal contours is proposed. This method maximizes correlation with cross-sectional geometry (r(2) = .998) and minimizes prediction errors in all cases. The results underscore the importance of accurate periosteal measurement in the quantification of bone strength, and suggest that, regardless of theoretical scaling predictions, external area based robusticity estimates involving the product of diaphyseal diameters are most directly comparable to cross-sectional geometric properties when they are standardized using the product of body mass and bone length.