Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification,Distraction Osteogenesis,or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens⁴. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice ^5-7, after application of bioactive molecules ^8,9, after altering physiological parameters of healing ¹⁰, after modifying the amount or time of stabilization ¹¹, after distraction osteogenesis ¹², after creation of a non-union ¹³, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.     

Metformin attenuates trauma‐induced heterotopic ossification via inhibition of Bone Morphogenetic Protein signalling

AMP‐activated protein kinase (AMPK) is an intracellular sensor of energy homoeostasis that is activated under energy stress and suppressed in energy surplus. AMPK activation leads to inhibition of anabolic processes that consume ATP. Osteogenic differentiation is a process that highly demands ATP during which AMPK is inhibited. The bone morphogenetic proteins (BMPs) signalling pathway plays an essential role in osteogenic differentiation. The present study examines the inhibitory effect of metformin on BMP signalling, osteogenic differentiation and trauma‐induced heterotopic ossification. Our results showed that metformin inhibited Smad1/5 phosphorylation induced by BMP6 in osteoblast MC3T3‐E1 cells, concurrent with up‐regulation of Smad6, and this effect was attenuated by knockdown of Smad6. Furthermore, we found that metformin suppressed ALP activity and mineralization of the cells, an event that was attenuated by the dominant negative mutant of AMPK and mimicked by its constitutively active mutant. Finally, administration of metformin prevented the trauma‐induced heterotopic ossification in mice. In conjuncture, AMPK activity and Smad6 and Smurf1 expression were enhanced by metformin treatment in the muscle of injured area, concurrently with the reduction of ALK2. Collectively, our study suggests that metformin prevents heterotopic ossification via activation of AMPK and subsequent up‐regulation of Smad6. Therefore, metformin could be a potential therapeutic drug for heterotopic ossification induced by traumatic injury.     

Heterotopic ossification of tendon and ligament

Much of the similarities of the tissue characteristics, pathologies and mechanisms of heterotopic ossification (HO) formation are shared between HO of tendon and ligament (HOTL). Unmet need and no effective treatment has been developed for HOTL, primarily attributable to poor understanding of cellular and molecular mechanisms. HOTL forms via endochondral ossification, a common process of most kinds of HO. HOTL is a dynamic pathologic process that includes trauma/injury, inflammation, mesenchymal stromal cell (MSC) recruitment, chondrogenic differentiation and, finally, ossification. A variety of signal pathways involve HOTL with multiple roles in different stages of HO formation, and here in this review, we summarize the progress and provide an up-to-date understanding of HOTL.     

PATTERNS OF OSSIFICATION IN SOUTHERN VERSUS NORTHERN PLACENTAL MAMMALS

Consensus on placental mammal phylogeny is fairly recent compared to that for vertebrates as a whole. A stable phylogenetic hypothesis enables investigation into the possibility that placental clades differ from one another in terms of their development. Here, we focus on the sequence of skeletal ossification as a possible source of developmental distinctiveness in “northern” (Laurasiatheria and Euarchontoglires) versus “southern” (Afrotheria and Xenarthra) placental clades. We contribute data on cranial and postcranial ossification events during growth in Afrotheria, including elephants, hyraxes, golden moles, tenrecs, sengis, and aardvarks. We use three different techniques to quantify sequence heterochrony: continuous method, sequence‐ANOVA (analysis of variance) and event‐paring/Parsimov. We show that afrotherians significantly differ from other placentals by an early ossification of the orbitosphenoid and caudal vertebrae. Our analysis also suggests that both southern placental groups show a greater degree of developmental variability; however, they rarely seem to vary in the same direction, especially regarding the shifts that differ statistically. The latter observation is inconsistent with the Atlantogenata hypothesis in which afrotherians are considered as the sister clade of xenarthrans. Interestingly, ancestral nodes for Laurasiatheria and Euarchontoglires show very similar trends and our results suggest that developmental homogeneity in some ossification sequences may be restricted to northern placental mammals (Boreoeutheria).     

Fetuin A is down-regulated in rats during heterotopic ossification after Achilles tenotomy

We investigated the role of fetuin A during heterotopic ossification (HO) in rats following Achilles tenotomy. We performed a right midpoint Achilles tenotomy on 24 rats. At 5 and 10 h after surgery, we investigated the formation of ectopic bone using X-ray and histological examination. We evaluated the mRNA level of fetuin A using real-time PCR. Presence of fetuin A in the Achilles tendon was assessed by immunohistochemical staining. We also measured the serum concentration of fetuin A using enzyme linked immunosorbent assay (ELISA). The expression of fetuin A was significantly decreased in both the liver and Achilles tendon during HO. ELISA showed a small amount of fetuin A in blood throughout the development of HO. Immunohistochemical staining showed that fetuin A was abundant in the ectopic bone. Fetuin A appears to be involved in the formation of ectopic bone induced by Achilles tenotomy, and a deficiency of fetuin A plays a role in the development of HO.     

Divergent responses of Atlantic cod to ocean acidification and food limitation

In order to understand the effect of global change on marine fishes, it is imperative to quantify the effects on fundamental parameters such as survival and growth. Larval survival and recruitment of the Atlantic cod (Gadus morhua) were found to be heavily impaired by end‐of‐century levels of ocean acidification. Here, we analysed larval growth among 35–36 days old surviving larvae, along with organ development and ossification of the skeleton. We combined CO₂ treatments (ambient: 503 µatm, elevated: 1,179 µatm) with food availability in order to evaluate the effect of energy limitation in addition to the ocean acidification stressor. As expected, larval size (as a proxy for growth) and skeletogenesis were positively affected by high food availability. We found significant interactions between acidification and food availability. Larvae fed ad libitum showed little difference in growth and skeletogenesis due to the CO₂ treatment. Larvae under energy limitation were significantly larger and had further developed skeletal structures in the elevated CO₂ treatment compared to the ambient CO₂ treatment. However, the elevated CO₂ group revealed impairments in critically important organs, such as the liver, and had comparatively smaller functional gills indicating a mismatch between size and function. It is therefore likely that individual larvae that had survived acidification treatments will suffer from impairments later during ontogeny. Our study highlights important allocation trade‐off between growth and organ development, which is critically important to interpret acidification effects on early life stages of fish.     

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.     

Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival

Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.     

A molecular analysis of matrix remodeling and angiogenesis during long bone development

The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a ‘molecular map’ of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.     

Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.