How is digit identity determined during limb development?

Digit identity has been studied using the chick embryo as a model system for more than 40 years. Using this model system, several milestone findings have been reported, such as the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), the Shh gene, and the theory of morphogen and positional information. These experimental results and models provided context for understanding pattern formation in developmental biology. The focus of this review is on the determination of digit identity during limb development. First, the history of studies on digit identity determination is described, followed by descriptions of the molecular mechanisms and current models for determination of digit identity. Finally, future questions and remarkable points will be discussed.

Development of the deep flexor tendons and lumbricalis muscle in the hand and foot: a histological study using human mid-term foetuses.

To revisit foetal development of the deep flexor tendons of the hand and foot, we examined the paraffin-embedded histology of 20 mid-term foetuses at 8-15 weeks of estimated gestational age (35-118 mm crown-rump length or CRL). At 8-9 weeks, in front of the metacarpal bones, the flexor pollicis longus and flexor digitorum profundus (FDP) muscles provided a plate-like, common tendon from which the lumbricalis muscles originated. However, in the foot, we had no evidence of such a common tendon. The flexor pollicis tendon was separated from the common tendon at 9-10 weeks possibly due to mechanical stress from the laterally growing thumb. Notably, at the lumbricalis muscle origins at 10-12 weeks, the FDP and flexor digitorum longus tendons remained undifferentiated and the primitive tenocytes were dispersed from them. The dispersed cells seemed to develop into an interface tissue between the lumbricalis muscle fibre and the deep tendon. In 3 of 5 specimens at 15 weeks, we found an excess number of the FDP tendons (5-7) in the proximal side of the lumbricalis muscle origin. However, the excess tendons dispersed in the lumbricalis muscle origin. The development of the lumbricalis muscle origin might follow the tendon splitting for four fingers. However, conversely, we hypothesised that the developing lumbricalis muscles re-arranged the deep flexor tendons to provide a configuration of one deep tendon per one finger (or toe). The quadrates plantae muscle seemed not to contribute on the re-arrangement.

A weighted osteon morphotype score outperforms regional osteon percent prevalence calculations for interpreting cortical bone adaptation.

Using circularly polarized light microscopy,we described a weighted-scoring method for quantifying regional distributions of six secondary osteon morphotypes(Skedros et al.: Bone 44 (2009) 392-403). This osteon morphotype score (MTS) strongly correlated with "tension" and "compression" cortices produced by habitual bending. In the present study, we hypothesized that the osteon MTS is superior to a relatively simpler method based on the percent prevalence (PP) of these osteon morphotypes. This was tested in proximal femoral diaphyses of adult chimpanzees and habitually bent bones: calcanei from sheep, deer, and horses, radii from sheep and horses, and third metacarpals (MC3s) from horses. Sheep tibiae were examined because their comparatively greater torsion/shear would not require regional variations in osteon morphotypes. Predominant collagen fiber orientation (CFO), a predictor of regionally prevalent/predominant strain mode, was quantified as image gray levels (birefringence). Ten PP calculations were conducted. Although PP calculations were similar to the osteon MTS in corroborating CFO differences between "tension" and "compression" cortices of the chimpanzee femora and most of the habitually bent bones, PP calculations failed to show a compression/tension difference in equine MC3s and sheep radii. With the exception of the prevalence of the "distributed" osteon morphotype, correlations of PP calculations with CFO were weak and/or negative. By contrast, the osteon MTS consistently showed positive correlations with predominant CFO. Compared with the osteon MTS and predominant CFO, regional variations in PP of osteon morpho types are not stronger predictors of nonuniform strain distributions produced by bending.

Two Cases of Intraosseous Pseudomyogenic (Epithelioid Sarcoma-Like) Hemangioendothelioma With Unusual Features, Expanding the Clinicopathological Spectrum.

Pseudomyogenic (epithelioid sarcoma-like) hemangioendothelioma (PMHE) is a distinct vascular neoplasm mostly observed in the lower extremities of young adults with characteristic histopathological features. In this article, we present 2 unusual cases of PMHE. Case 1: A 28-year-old male presented with pain and stiffness in his left shoulder. Radiologic examination revealed an expansile, lytic, heterogeneously enhancing, destructive lesion in his left scapula, along with multiple avid marrow lesions in his other bones. Biopsy revealed a cellular tumor composed of plump spindly and epithelioid cells arranged in fascicles and focally, in clusters, admixed with neutrophils and histiocytes, with interspersed many osteoclast-like giant cells and reactive bone. Case 2: A 63-year-old male presented pain and swelling in his right middle finger, with no other lesion elsewhere in his body. Radiologic imaging disclosed a 1.5-cm-sized lobulated, expansile, lytic, destructive lesion in the distal end of the third metacarpal bone of his right hand. Biopsy examination revealed a cellular spindle cell tumor, composed of plump spindly cells, arranged in fascicles, including "tadpole-like" or "strap-like" cells and interspersed inflammatory cells. Immunohistochemically, tumor cells in both cases were positive for AE1/AE3, CD31, and Fli1, while negative for desmin and CD34. INI11 was retained. The presented cases of PMHE, occurring at unusual locations, in an older individual in the second case, along with variable histopathological features, noted in the first case, seem to expand the clinicopathological spectrum of these uncommon neoplasms. Differential diagnoses and review of similar cases are presented.

The relationship between microstructure, stiffness and compressive fatigue life of equine subchondral bone.

Subchondral bone injuries often precede articular cartilage damage in osteoarthritis and are common in thoroughbred racehorses due to the accumulation of fatigue damage from high speed racing and training. Thus, racehorses provide a model to investigate the role of subchondral bone in joint disease. We assessed the association of horse and racing related factors and micro-CT based micromorphology of three separate subchondral bone layers with the initial stiffness and compressive fatigue life of bone plugs. Furthermore, we investigated three different definitions of fatigue failure of subchondral bone during compressive fatigue testing. Initial stiffness was 2,362 ±â€¯443 MPa (mean ±â€¯standard deviation). Median compressive fatigue life during cyclic loading to -78 MPa was 16,879 (range 210 to 57,064). Subchondral bone stiffness increased over a median of 24% (range 3%-42%) of fatigue life to a maximum of 3,614 ±â€¯635 MPa. Compressive fatigue life was positively associated with bone volume fraction in the deeper layers of subchondral bone, maximal stiffness, and the number of cycles to maximal stiffness. Initial stiffness was positively associated with tissue mineral density in the deeper layers and bone volume fraction in the superficial layer. Most specimens with a fatigue life of less than 5,500 cycles fractured grossly before reaching 30% reduction of maximal stiffness. Cycles to 10% reduction of maximal stiffness correlated strongly with cycles to lowest recorded stiffness at gross fracture and thus is a valid alternative failure definition for compressive fatigue testing of subchondral bone. Our results show that subchondral bone sclerosis as a result of high speed exercise and measured as bone volume fraction is positively associated with compressive fatigue life and thus has a protective effect on subchondral bone. Further research is required to reconcile this finding with the common collocation of fatigue damage in sclerotic subchondral bone of racehorses.

Chondrocyte deformation induces mitochondrial distortion and heterogeneous intracellular strain fields.

Chondrocyte mechanotransduction is poorly understood but may involve cell deformation and associated distortion of intracellular structures and organelles. This study quantifies the intracellular displacement and strain fields associated with chondrocyte deformation and in particular the distortion of the mitochondria network, which may have a role in mechanotransduction. Isolated articular chondrocytes were compressed in agarose constructs and simultaneously visualised using confocal microscopy. An optimised digital image correlation technique was developed to calculate the local intracellular displacement and strain fields using confocal images of fluorescently labelled mitochondria. The mitochondria formed a dynamic fibrous network or reticulum, which co-localised with microtubules and vimentin intermediate filaments. Cell deformation induced distortion of the mitochondria, which collapsed in the axis of compression with a resulting loss of volume. Compression generated heterogeneous intracellular strain fields indicating mechanical heterogeneity within the cytoplasm. The study provides evidence supporting the potential involvement of mitochondrial deformation in chondrocyte mechanotransduction, possibly involving strain-mediated release of reactive oxygen species. Furthermore the heterogeneous strain fields, which appear to be influenced by intracellular structure and organisation, may generate significant heterogeneity in mechanotransduction behaviour for cells subjected to identical levels of deformation.

Integrin-mediated mechanotransduction in IL-1 beta stimulated chondrocytes.

Mechanical loading and interleukin-1 beta (IL-1 beta) influence the release of nitric oxide (*NO) and prostaglandin E2 (PGE2) from articular chondrocytes via distinct signalling mechanisms. The exact nature of the interplay between the respective signalling pathways remains unclear. Recent studies have shown that integrins act as mechanoreceptors and may transduce extracellular stimuli into intracellular signals, thereby influencing cellular response. The current study demonstrates that the application of dynamic compression induced an inhibition of *NO and an upregulation of cell proliferation and proteoglycan synthesis in the presence and absence of IL-1 beta. PGE2 release was not affected by dynamic compression in the absence of IL-1 beta but was inhibited in the presence of the cytokine. The integrin binding peptide, GRGDSP, abolished or reversed the compression-induced alterations in all four parameters assessed in the presence and absence of IL-1 beta. The non-binding control peptide, GRADSP, had no effect. These data clearly demonstrate that the metabolic response of the chondrocytes to dynamic compression in the presence and absence of IL-1 beta, are integrin mediated.

Cortical bone response to the presence of load-bearing percutaneous osseointegrated prostheses.

Although the current percutaneous osseointegrated (OI) prosthetic attachment systems are novel clinical treatments for patients with limb loss, there have only been limited translational studies undertaken to date. To bridge this knowledge gap, from a larger study group of 86 animals that were implanted with a novel percutaneous OI implant construct, 33 sheep were randomly selected from the 0-, 3-, 6-, 9- and 12-month groups for histomorphometric analyses of periprosthetic cortical bone tissue. At necropsy, implanted and nonimplanted limbs were harvested and processed for the evaluation of cortical bone porosity and mineral apposition rate (MAR). The data showed a maximum increase in bone porosity within the first 3 months following implantation and then a progressive reduction in porosity to the baseline steady-state ("Time 0") value by 12 months. The data further verified that the MAR increased during the first 6 months of implantation, reaching a plateau between 6 and 9 months, followed by a progressive decline to the baseline steady state. It was concluded that clinical load bearing and falls precautions, taken during the first 3-6 months following percutaneous OI device implantation surgery, could greatly limit bone fractures during this vulnerable time of increasing cortical bone porosity.

Interpreting cortical bone adaptation and load history by quantifying osteon morphotypes in circularly polarized light images.

Birefringence variations in circularly polarized light (CPL) images of thin plane-parallel sections of cortical bone can be used to quantify regional differences in predominant collagen fiber orientation (CFO). Using CPL images of equine third metacarpals (MC3s), R.B. Martin, V.A. Gibson, S.M. Stover, J.C. Gibeling, and L.V. Griffin. (40) described six secondary osteon variants ('morphotypes') and suggested that differences in their regional prevalence affect fatigue resistance and toughness. They devised a numerical osteon morphotype score (MTS) for quantifying regional differences in osteon morphotypes. We have observed that a modification of this score could significantly improve its use for interpreting load history. We hypothesized that our modified osteon MTS would more accurately reveal differences in osteon MTSs between opposing "tension" and "compression" cortices of diaphyses of habitually bent bones. This was tested using CPL images in transverse sections of calcanei from sheep, deer, and horses, and radii from sheep and horses. Equine MC3s and sheep tibiae were examined as controls because they experience comparatively greater load complexity that, because of increased prevalence of torsion/shear, would not require regional mechanical enhancements provided by different osteon morphotypes. Predominant CFO, which can reliably reflect adaptation for a regionally prevalent strain mode, was quantified as mean gray levels from birefringence of entire images (excluding pore spaces) in anterior, posterior, medial, and lateral cortices. Results showed that, in contrast to the original scoring scheme of Martin et al., the modified scheme revealed significant anterior/posterior differences in osteon MTSs in nearly all "tension/compression" bones (p<0.0001), but not in equine MC3s (p=0.30) and sheep tibiae (p=0.35). Among habitually bent bones, sheep radii were the exception; relatively lower osteon populations and the birefringence of the primary bone contributed to this result. Correlations between osteon MTSs using the scoring scheme of Martin et al. with CFO data from all regions of each bone invariably demonstrated weak-to-moderate negative correlations. This contrasts with typically high positive correlations between modified osteon MTSs and regional CFO. These results show that the modified osteon MTS can be a strong correlate of predominant CFO and of the non-uniform strain distribution produced by habitual bending.