Restraint upon Embryonic Metatarsal Ex Vivo Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway.

Mechanical cues play a vital role in limb skeletal development, yet their influence and underpinning mechanisms in the regulation of endochondral ossification (EO) processes are incompletely defined. Furthermore, interactions between endochondral growth and mechanics and the mTOR/NF-ĸB pathways are yet to be explored. An appreciation of how mechanical cues regulate EO would also clearly be beneficial in the context of fracture healing and bone diseases, where these processes are recapitulated. The study herein addresses the hypothesis that the mTOR/NF-ĸB pathways interact with mechanics to control endochondral growth. To test this, murine embryonic metatarsals were incubated ex vivo in a hydrogel, allowing for the effects of quasi-static loading on longitudinal growth to be assessed. The results showed significant restriction of metatarsal growth under quasi-static loading during a 14-day period and concentration-dependent sensitivity to hydrogel-related restriction. This study also showed that hydrogel-treated metatarsals retain their viability and do not present with increased apoptosis. Metatarsals exhibited reversal of the growth-restriction when co-incubated with mTOR compounds, whilst it was found that these compounds showed no effects under basal culture conditions. Transcriptional changes linked to endochondral growth were assessed and downregulation of Col2 and Acan was observed in hydrogel-treated metatarsi at day 7. Furthermore, cell cycle analyses confirmed the presence of chondrocytes exhibiting S-G2/M arrest. These data indicate that quasi-static load provokes chondrocyte cell cycle arrest, which is partly overcome by mTOR, with a less marked interaction for NF-ĸB regulators.

Radial shock waves prevent growth retardation caused by the clinically used drug vismodegib in ex vivo cultured bones.

In childhood medulloblastoma patients, the hedgehog antagonist vismodegib is an effective anti-cancer treatment but unfortunately induces irreversible growth arrests and growth impairment limiting its use in skeletally immature patients. We hypothesized that radial shock wave treatment (rSWT) may protect drug-induced growth impairment owing to its osteogenic effects. Fetal rat metatarsal bones were exposed to vismodegib (day 0-5; 100 nM) and/or rSWT (single session); other bones from day 1 were continuously exposed to a Gli1 antagonist (GANT61; 10 µM) and/or rSWT (single session). Control bones were untreated. The bone length was measured at intervals; histomorphometric analysis and immunostaining for PCNA, Gli1, and Ihh were performed on the sectioned bones. Bones treated with vismodegib showed impaired bone growth, reduced height of the resting-proliferative zone and reduced hypertrophic cell size compared to control. In vismodegib treated bones, a single session of rSWT partially rescued bone growth, increased the growth velocity, hypertrophic cell size, and restored growth plate morphology. Bones exposed to GANT61 showed impaired bone growth and disorganized growth plate while when combined with rSWT these effects were partially prevented. Locally applied rSWT had a chondroprotective effect in rat metatarsal bones and suggest a novel strategy to prevent growth impairment caused by vismodegib.

Radial Extracorporeal Shock Wave Treatment Promotes Bone Growth and Chondrogenesis in Cultured Fetal Rat Metatarsal Bones.

BACKGROUND: Substantial evidence exists to show the positive effects of radialextracorporeal shock wave therapy (ESWT) on bone formation. However, it is unknown whether rESWT can act locally at the growth plate level to stimulate linear bone growth. One way to achieve this is to stimulate chondrogenesis in the growth plate without depending on circulating systemic growth factors. We wished to see whether rESWT would stimulate metatarsal rat growth plates in the absence of vascularity and associated systemic growth factors. QUESTIONS/PURPOSES: To study the direct effects of rESWT on growth plate chondrogenesis, we asked: (1) Does rESWT stimulate longitudinal bone growth of ex vivo cultured bones? (2) Does rESWT cause any morphological changes in the growth plate? (3) Does rESWT locally activate proteins specific to growth plate chondrogenesis? METHODS: Metatarsal bones from rat fetuses were untreated (controls: n = 15) or exposed to a single application of rESWT at a low dose (500 impulses, 5 Hz, 90 mJ; n = 15), mid-dose (500 impulses, 5 Hz, 120 mJ; n = 14) or high dose (500 impulses, 10 Hz, 180 mJ; n = 34) and cultured for 14 days. Bone lengths were measured on Days 0, 4, 7, and 14. After 14 days of culturing, growth plate morphology was assessed with a histomorphometric analysis in which hypertrophic cell size (> 7 µm) and hypertrophic zone height were measured (n = 6 bones each). Immunostaining for specific regulatory proteins involved in chondrogenesis and corresponding staining were quantitated digitally by a single observer using the automated threshold method in ImageJ software (n = 6 bones per group). A p value < 0.05 indicated a significant difference. RESULTS: The bone length in the high-dose rESWT group was increased compared with that in untreated controls (4.46 mm ± 0.75 mm; 95% confidence interval, 3.28-3.71 and control: 3.50 mm ± 0.38 mm; 95% CI, 4.19-4.72; p = 0.01). Mechanistic studies of the growth plate's cartilage revealed that high-dose rESWT increased the number of proliferative chondrocytes compared with untreated control bones (1363 ± 393 immunopositive cells per bone and 500 ± 413 immunopositive cells per bone, respectively; p = 0.04) and increased the diameter of hypertrophic chondrocytes (18 ± 3 µm and 13 ± 3 µm, respectively; p < 0.001). This was accompanied by activation of insulin-like growth factor-1 (1015 ± 322 immunopositive cells per bone and 270 ± 121 immunopositive cells per bone, respectively; p = 0.043) and nuclear factor-kappa beta signaling (1029 ± 262 immunopositive cells per bone and 350 ± 60 immunopositive cells per bone, respectively; p = 0.01) and increased levels of the anti-apoptotic proteins B-cell lymphoma 2 (718 ± 86 immunopositive cells per bone and 35 ± 11 immunopositive cells per bone, respectively; p < 0.001) and B-cell lymphoma-extra-large (107 ± 7 immunopositive cells per bone and 34 ± 6 immunopositive cells per bone, respectively; p < 0.001). CONCLUSION: In a model of cultured fetal rat metatarsals, rESWT increased longitudinal bone growth by locally inducing chondrogenesis. To verify whether rESWT can also stimulate bone growth in the presence of systemic circulatory factors, further studies are needed. CLINICAL RELEVANCE: This preclinical proof-of-concept study shows that high-dose rESWT can stimulate longitudinal bone growth and growth plate chondrogenesis in cultured fetal rat metatarsal bones. A confirmatory in vivo study in skeletally immature animals must be performed before any clinical studies.

Analysis of Serial Radiographs of the Foot to Determine Normative Values for the Growth of the First Metatarsal to Guide Hemiepiphysiodesis for Immature Hallux Valgus.

BACKGROUND: Hallux valgus deformity in the immature patient can be difficult to manage, as osteotomy can result in recurrence with additional growth. Lateral hemiepiphysiodesis of the first metatarsal offers a promising alternative, by permitting gradual correction of the intermetatarsal angle with growth. An important limitation of this approach is the lack of normative tables of first metatarsal growth to guide timing of intervention. METHODS: First metatarsal lengths were measured from anteroposterior foot radiographs of children. For females, 95 patients totaling 894 radiographs were used ranging from 6 months to 18 years of age. For males, 122 patients totaling 1018 radiographs were measured ranging from 8 months to 19.5 years of age. All patients with image series including a closed proximal metatarsal physis were sorted into an older group, with multipliers generated by setting last image to a multiplier of 1. Patients with serial imaging not inclusive of a closed physis were classified as a younger group, with multipliers based off of the multiplier at age 7 from the older group. First metatarsal multiplier values were then compared with published multiplier values for the overall foot. RESULTS: For both females and males, the multipliers followed a logarithmic curve versus age, with R values of 0.921 and 0.888, respectively. Comparison of the first metatarsal multiplier values with previously studied multiplier values of the entire foot showed high correlation with ICC=0.955 for females and ICC=0.969 for males. CONCLUSIONS: The pattern of growth of the first metatarsal follows a logarithmic regression curve. These normative tables allow for clinical prediction of first metatarsal remaining growth based on age and sex, and in turn guide timing of hemiepiphysiodesis for the surgical correction of hallux valgus deformity. CLINICAL RELEVANCE: The normative tables generated in this study can be used for the calculation of hemiepiphysiodesis and the timing of intervention. Future clinical correlation studies will be important.

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

Ageing Cattle: The Use of Radiographic Examinations on Cattle Metapodials from Eketorp Ringfort on the Island of Öland in Sweden.

In this paper conventional X-ray analysis of cattle metapodials is used to study the age structure of slaughtered cattle at Eketorp ringfort on the island of Öland, Sweden. The X-ray analyses suggest that several animals in both phases were slaughtered aged 4-8 years. More oxen/bulls than cows reached the advanced age of over 8 years, yet in phase III more oxen/bulls seem to have been slaughtered between the ages of 2 and 8 years. These differences may reflect a change in demand for meat related to the character of the site. The results also show a correlation between metapodials with a pathology connected to biomechanical stress and older animals. This suggests that male cattle were used both in meat production and as draught animals. Asymmetry in male metatarsals such as distal broadening of the lateral part of the medial trochlea was visible on the X-ray images. The bone element also indicates a denser outer cortex of the medial diaphysis in comparison to the inner medulla. This could be the result of repetitive mechanical stress. Two metatarsals from cows were documented with distal asymmetry indicating that cows were also used as working animals. Bone elements with changes in the articular surfaces were more common in metapodials from cows with an X-ray age of over 3-4 years. These results highlighted the slaughter age difference between oxen/bulls and cows, enabling a better understanding of animal husbandry and the selection of draught cattle at Eketorp ringfort.

Increased linear bone growth by GH in the absence of SOCS2 is independent of IGF-1.

Growth hormone (GH) signaling is essential for postnatal linear bone growth, but the relative importance of GHs actions on the liver and/or growth plate cartilage remains unclear. The importance of liver derived insulin like-growth factor-1 (IGF-1) for endochondral growth has recently been challenged. Here, we investigate linear growth in Suppressor of Cytokine Signaling-2 (SOCS2) knockout mice, which have enhanced growth despite normal systemic GH/IGF-1 levels. Wild-type embryonic ex vivo metatarsals failed to exhibit increased linear growth in response to GH, but displayed increased Socs2 transcript levels (P < 0.01). In the absence of SOCS2, GH treatment enhanced metatarsal linear growth over a 12 day period. Despite this increase, IGF-1 transcript and protein levels were not increased in response to GH. In accordance with these data, IGF-1 levels were unchanged in GH-challenged postnatal Socs2(-/-) conditioned medium despite metatarsals showing enhanced linear growth. Growth-plate Igf1 mRNA levels were not elevated in juvenile Socs2(-/-) mice. GH did however elevate IGF-binding protein 3 levels in conditioned medium from GH challenged metatarsals and this was more apparent in Socs2(-/-) metatarsals. GH did not enhance the growth of Socs2(-/-) metatarsals when the IGF receptor was inhibited, suggesting that IGF receptor mediated mechanisms are required. IGF-2 may be responsible as IGF-2 promoted metatarsal growth and Igf2 expression was elevated in Socs2(-/-) (but not WT) metatarsals in response to GH. These studies emphasise the critical importance of SOCS2 in regulating GHs ability to promote bone growth. Also, GH appears to act directly on the metatarsals of Socs2(-/-) mice, promoting growth via a mechanism that is independent of IGF-1.

Identification of receptor-type protein tyrosine phosphatase µ as a new marker for osteocytes.

Osteocytes are the predominant cells in bone, where they form a cellular network and display important functions in bone homeostasis, phosphate metabolism and mechanical transduction. Several proteins strongly expressed by osteocytes are involved in these processes, e.g., sclerostin, DMP-1, PHEX, FGF23 and MEPE, while others are upregulated during differentiation of osteoblasts into osteocytes, e.g., osteocalcin and E11. The receptor-type protein tyrosine phosphatase µ (RPTPµ) has been described to be expressed in cells which display a cellular network, e.g., endothelial and neuronal cells, and is implied in mechanotransduction. In a capillary outgrowth assay using metatarsals derived from RPTPµ-knock-out/LacZ knock-in mice, we observed that the capillary structures grown out of the metatarsals were stained blue, as expected. Surprisingly, cells within the metatarsal bone tissue were positive for LacZ activity as well, indicating that RPTPµ is also expressed by osteocytes. Subsequent histochemical analysis showed that within bone, RPTPµ is expressed exclusively in early-stage osteocytes. Analysis of bone marrow cell cultures revealed that osteocytes are present in the nodules and an enzymatic assay enabled the quantification of the amount of osteocytes. No apparent bone phenotype was observed when tibiae of RPTPµ-knock-out/LacZ knock-in mice were analyzed by µCT at several time points during aging, although a significant reduction in cortical bone was observed in RPTPµ-knock-out/LacZ knock-in mice at 20 weeks. Changes in trabecular bone were more subtle. Our data show that RPTPµ is a new marker for osteocytes.

Crecimiento longitudinal posnatal del primer metatarsiano / Longitudinal postnatal growth of the first metatarsal bone

Introducción: Si bien el crecimiento prenatal y posnatal del pie ha sido documentado hace varios años, el crecimiento longitudinal del primer metatarsiano en particular, no ha sido estudiado previamente. El objetivo del estudio es determinar el patrón de crecimiento longitudinal posnatal de este hueso y compararlo con el del pie y los huesos largos del miembro inferior. Materiales y Métodos: Mediante una búsqueda informatizada, se identificaron pacientes <18 años de edad con radiografías informadas como “normal” por el radiólogo. Se analizó una muestra de 886 pacientes divididos en 18 grupos según la edad (0-11 meses, 1 año, 2 años, etc.) y el sexo. El análisis de las imágenes se realizó con un software de imágenes Kodak Carestream PACS. Resultados: El largo promedio en el primer grupo fue de 19,91 mm (3,20; 15,22-25,62). El largo promedio en el último grupo fue de 66,13 mm (5,33; 52,50-77,18). La tasa de crecimiento anual fue de 2,71 mm. La edad promedio al momento del cierre de la fisis fue de 14.85 años (± 1.64) para los varones y 14.77 años (± 3.63) para las niñas. Conclusión: El crecimiento del primer metatarsiano acompaña el crecimiento longitudinal del pie, pero no el de los huesos largos del miembro inferior. Las curvas de crecimiento del primer metatarsiano descritas en este artículo pueden ser aplicadas en patologías que afectan el desarrollo del pie o que requieren cirugía de corrección sobre el primer metatarsiano, o se las puede emplear como estándar de referencia en futuros estudios. Nivel de Evidencia: III.

Background: While prenatal and postnatal growth of the foot has been documented several years ago, longitudinal growth of the first metatarsal has not been previously evaluated. The aim of the study is to determine the postnatal longitudinal growth pattern of this bone and compare it with the foot and lower limb long bones. Methods: Through a computerized image search, we identified patients <18 years old with radiographs reported as “normal” by the radiologist. A sample of 886 patients was divided into 18 groups according to age (0-11 months, 1 year, 2 years, etc.) and sex. Analysis was performed using Kodak Carestream imaging software PACS. Results: The average length in the first group was 19.91 mm (3.20, 15.22-25.62). The average length in the last group was 66.13 mm (5.33, 52.50-77.18). Annual growth rate was 2.71 mm. The average age at the time of physeal closure was 14.85 years (± 1.64) for boys and 14.77 years (± 3.63) for girls. Conclusion: Longitudinal growth of the first metatarsal mimics the growth of foot but not that of the long bones of the lower limb. Growth curves described in this article can be applied to conditions that affect foot development or require corrective surgery on the first metatarsal, as well as a standard reference in future studies. Level of Evidence: III.

The utility of the proximal epiphysis of the fifth metatarsal in age estimation.

Radiographs of 277 living individuals were assessed via a numerical scoring system to determine the timing of appearance and degree of fusion between the proximal epiphysis of the fifth metatarsal and its diaphysis. The epiphysis was observed to first appear in females at 8 years and 10 years in males and fuse by 14 years in females and 15 years in males. When assessing the level of agreement of category assignment, inter-observer agreement was 78% for females and 64% for males whereas intra-observer agreement was 77% for females and 86.1% for males. These results suggest that the maturation of the proximal epiphysis of the fifth metatarsal may be of value in age estimation in the child and that the scoring system is sufficiently robust to merit continued investigation. Previously this epiphysis has been considered an inconstant feature, but this research confirmed its presence in all individuals studied.

[Radiologic study on postnatal ossification of the first metatarsal bone]. / Estudio radiológico sobre la osificación postnatal del primer metatarsiano.

BACKGROUND: Classical descriptions of the ossification of the first metatarsal bone show the existence of a single proximal secondary ossification nucleus, mimicking the typical ossification of a phalanx. However, there are studies that show the presence of a second distal ossification nucleus and discuss its nature: epiphysis or pseudoepiphysis. The objective of the study is to establish the prevalence of such distal nucleus, determine its role in the growth of the radius and its relation with different qualitative variables. MATERIAL AND METHODS: We conducted a retrospective study that included 971 dorsoplantar images from 225 patients. We analyzed the presence of such nucleus, the age of appearance and closure, the relation with different qualitative variables and their contribution to the longitudinal growth of the first metatarsal bone. RESULTS: The distal ossification nucleus appears in 40% of all images and in 81.1% of cases ages 4-7 years. Mean age of appearance is 3.07 years, while physeal closure occurs at a mean of 7.67 years. We found a significantly higher frequency of physeal closure in children, patients with Egyptian type foot, flat foot, and index-plus and Egyptian type foot combinations. The definitive length of the first metatarsal bone is greater in patients with a distal ossification nucleus. CONCLUSIONS: We may state the presence of the distal ossification nucleus of the first metatarsal bone that behaves as a fertile growth cartilage.

Bortezomib is cytotoxic to the human growth plate and permanently impairs bone growth in young mice.

Bortezomib, a novel proteasome inhibitor approved for the treatment of cancer in adults, has recently been introduced in pediatric clinical trials. Any tissue-specific side effects on bone development have to our knowledge not yet been explored. To address this, we experimentally studied the effects of bortezomib in vivo in young mice and in vitro in organ cultures of rat metatarsal bones and human growth plate cartilage, as well as in a rat chondrocytic cell line. We found that bortezomib while efficiently blocking the ubiquitin/proteasome system (UPS) caused significant growth impairment in mice, by increasing resting/stem-like chondrocyte apoptosis. Our data support a local action of bortezomib, directly targeting growth plate chondrocytes leading to decreased bone growth since no suppression of serum levels of insulin-like growth factor-I (IGF-I) was observed. A local effect of bortezomib was confirmed in cultured rat metatarsal bones where bortezomib efficiently caused growth retardation in a dose dependent and irreversible manner, an effect linked to increased chondrocyte apoptosis, mainly of resting/stem-like chondrocytes. The cytotoxicity of bortezomib was also evaluated in a unique model of cultured human growth plate cartilage, which was found to be highly sensitive to bortezomib. Mechanistic studies of apoptotic pathways indicated that bortezomib induced activation of p53 and Bax, as well as cleavage of caspases and poly-ADP-ribose polymerase (PARP) in exposed chondrocytes. Our observations, confirmed in vivo and in vitro, suggest that bone growth could potentially be suppressed in children treated with bortezomib. We therefore propose that longitudinal bone growth should be closely monitored in ongoing clinical pediatric trials of this promising anti-cancer drug.

Pro-inflammatory cytokines produced by growth plate chondrocytes may act locally to modulate longitudinal bone growth.

INTRODUCTION: Interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α), both cytokines upregulated during chronic inflammation, are known to suppress bone growth. So far no role of these cytokines in modulation of normal bone growth has been established. METHODOLOGY: Applying RT-PCR and immunohistochemistry, expression of IL-1ß and TNF-α was studied in cultured fetal (E20) rat metatarsal bones. Anakinra (500 µg/ml; IL-1 receptor antagonist) and/or etanercept (500 µg/ml; soluble TNF-α receptor) were used to block cytokine actions. RESULTS: The local expression of IL-1ß and TNF-α was confirmed in the rat metatarsal growth plate. When cultured for 12 days and compared to control, the length of bones exposed to anakinra, etanercept, or anakinra plus etanercept increased by 7.7 ± 2.0 (p < 0.05), 11.7 ± 2.8 (p < 0.01) and 20.3 ± 1.9% (p < 0.001), respectively, while the height of the hypertrophic growth plate zone (collagen X staining) increased by 11.0 ± 6.7, 17.4 ± 7.1 and 43.1 ± 5.0% (p < 0.01), respectively. Moreover, etanercept increased chondrocyte proliferation (BrdU incorporation). CONCLUSION: Our findings that IL-1ß and TNF-α are produced by growth plate chondrocytes and that their antagonists improve growth of cultured metatarsal bones suggest that these cytokines play a physiological role in the normal regulation of longitudinal bone growth.

Congenic mice provide in vivo evidence for a genetic locus that modulates intrinsic transforming growth factor ß1-mediated signaling and bone acquisition.

Osteoporosis, the most common skeletal disorder, is characterized by low bone mineral density (BMD) and an increased risk of fragility fractures. BMD is the best clinical predictor of future osteoporotic fracture risk, but is a complex trait controlled by multiple environmental and genetic determinants with individually modest effects. Quantitative trait locus (QTL) mapping is a powerful method for identifying chromosomal regions encompassing genes involved in shaping complex phenotypes, such as BMD. Here we have applied QTL analysis to male and female genetically-heterogeneous F(2) mice derived from a cross between C57BL/6 and DBA/2 strains, and have identified 11 loci contributing to femoral BMD. Further analysis of a QTL on mouse chromosome 7 following the generation of reciprocal congenic strains has allowed us to determine that the high BMD trait, which tracks with the DBA/2 chromosome and exerts equivalent effects on male and female mice, is manifested by enhanced osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and by increased growth of metatarsal bones in short-term primary culture. An insertion/deletion DNA polymorphism in Ltbp4 exon 12 that causes the in-frame removal of 12 codons in the DBA/2-derived gene maps within 0.6 Mb of the marker most tightly linked to the QTL. LTBP4, one of four paralogous mouse proteins that modify the bioavailability of the transforming growth factor ß (TGF-ß) family of growth factors, is expressed in differentiating MSC-derived osteoblasts and in long bones, and reduced responsiveness to TGF-ß1 is observed in MSCs of mice homozygous for the DBA/2 chromosome 7. Taken together, our results identify a potential genetic and biochemical relationship between decreased TGF-ß1-mediated signaling and enhanced femoral BMD that may be regulated by a variant LTBP4 molecule.

Vinculin functions as regulator of chondrogenesis.

To identify the genes involved in chondrocytic differentiation, we applied gene trap mutagenesis to a murine mesenchymal chondrogenic cell line ATDC5 and isolated a clone in which the gene encoding vinculin was trapped. The trapped allele was assumed to express a fusion protein containing a truncated vinculin lacking the tail domain and the geo product derived from the trap vector. The truncated vinculin was suggested to exert a dominant negative effect. Impaired functioning of vinculin caused by gene trapping in ATDC5 cells or knockdown in primary chondrocytes resulted in the reduced expression of chondrocyte-specific genes, including Col2a1, aggrecan, and Col10a1. The expression of Runx2 also was suppressed by the dysfunctional vinculin. On the other hand, the expression of Sox9, encoding a key transcription factor for chondrogenesis, was retained. Knockdown of vinculin in metatarsal organ cultures impaired the growth of the explants and reduced the expression of Col2a1 and aggrecan. Gene trapping or knockdown of vinculin decreased the phosphorylation of ERK1/2 but increased that of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) and Akt during chondrocytic differentiation, suggesting a disturbance of signaling by insulin-like growth factor I (IGF-I). Knockdown of vinculin in the metatarsal organ culture abrogated the IGF-I-induced growth and inhibited the up-regulation of Col2a1 and aggrecan expression by IGF-I. Loss of vinculin function in differentiating chondrocytes impaired the activation of the p38 MAPK pathway also, suggesting its involvement in the regulation of chondrogenesis by vinculin. Our results indicate a tissue-specific function of vinculin in cartilage whereby it controls chondrocytic differentiation.

The evolution of mammalian body temperature: the Cenozoic supraendothermic pulses.

In this study, I investigated the source(s) of variation in the body temperatures of mammals. I also attempted to reconstruct ancestral normothermic rest-phase body temperature states using a maximum parsimony approach. Body temperature at the familial level is not correlated with body mass. For small mammals, except the Macroscelidae, previously identified correlates, such as climate adaptation and zoogeography explained some, but not all, T(b) apomorphies. At the species level in large cursorial mammals, there was a significant correlation between body temperature and the ratio between metatarsal length and femur length, the proxy for stride length and cursoriality. With the exception of two primate families, all supraendothermic (T(b) > 37.9°C) mammals are cursorial, including Artiodactyla, Lagomorpha, some large Rodentia, and Carnivora. The ruminant supraendothermic cursorial pulse is putatively associated with global cooling and vegetation changes following the Paleocene-Eocene Thermal Maximum. Reconstructed ancestral body temperatures were highly unrealistic deep within the mammalian phylogeny because of the lack of fossil T(b) data that effectively creates ghost lineages. However, it is anticipated that the method of estimating body temperature from the abundance of ¹³C-¹8O bonds in the carbonate component of tooth bioapatite in both extant and extinct animals may be a very promising tool for estimating the T(b) of extinct mammals. Fossil T(b) data are essential for discerning derived T(b) reversals from ancestral states, and verifying the dates of supraendothermic pulses.

SOCS2 is the critical regulator of GH action in murine growth plate chondrogenesis.

Suppressor of Cytokine Signaling-2 (SOCS2) is a negative regulator of growth hormone (GH) signaling and bone growth via inhibition of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. This has been classically demonstrated by the overgrowth phenotype of SOCS2(-/-) mice, which has normal systemic insulin-like growth factor 1 (IGF-1) levels. The local effects of GH on bone growth are equivocal, and therefore this study aimed to understand better the SOCS2 signaling mechanisms mediating the local actions of GH on epiphyseal chondrocytes and bone growth. SOCS2, in contrast to SOCS1 and SOCS3 expression, was increased in cultured chondrocytes after GH challenge. Gain- and loss-of-function studies indicated that GH-stimulated chondrocyte STATs-1, -3, and -5 phosphorylation was increased in SOCS2(-/-) chondrocytes but not in cells overexpressing SOCS2. This increased chondrocyte STAT signaling in the absence of SOCS2 is likely to explain the observed GH stimulation of longitudinal growth of cultured SOCS2(-/-) embryonic metatarsals and the proliferation of chondrocytes within. Consistent with this metatarsal data, bone growth rates, growth plate widths, and chondrocyte proliferation were all increased in SOCS2(-/-) 6-week-old mice as was the number of phosphorylated STAT-5-positive hypertrophic chondrocytes. The SOCS2(-/-) mouse represents a valid model for studying the local effects of GH on bone growth.

Proximal epiphysis of the second metatarsal: normal trait, possible contribution to growth, and clinical implications.

Analysis of 998 dorsoplantar foot radiographs of 231 patients was performed. Age ranged between 0.21 and 19.94 years. The authors proved the existence of a previously nondescribed additional proximal ossification center in the second metatarsal. This additional proximal center was seen in 78 of 998 radiographs, which corresponds with 7 of the 231 patients. If only 111 patients within the age range in which the additional proximal center appears are considered, the true prevalence of the additional proximal center was found in 7 (6.3%) patients. This additional proximal center was found to be bilateral in 4 (57.1%) of these 7 patients. The presence of the proximal center was more common in girls and was associated with index plus metatarsal type (P=.000) and foot pathology, especially flatfoot (P=.000). Age of appearance was 2.3 ± 0.24 years and age of fusion was 5.5 ± 2.2 years. Using a descriptive maturation scale, the authors classified the ossification stages of the additional proximal center in 4 stages: rudimentary, fully formed, partially fused, and fully fused. After creating a mathematical ratio, the authors could not prove that the presence of this additional proximal center increased the length of the second metatarsal or the complete second ray.

The biologics anakinra and etanercept prevent cytokine-induced growth retardation in cultured fetal rat metatarsal bones.

BACKGROUND/AIMS: Chronic inflammation during childhood often leads to impaired bone growth and reduced adult height. Proinflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF)-α synergistically impair bone growth in vitro. We hypothesized that biologic agents may rescue bones from cytokine-induced growth impairment and that insulin growth factor (IGF)-I may potentiate such an effect. METHODOLOGY: Metatarsal bones from fetal Sprague-Dawley rats (19-20 days p.c.) were treated with IL-1ß plus TNF-α, or the combination of these cytokines with anakinra (IL-1 receptor antagonist), etanercept (TNF-inhibitor) and/or IGF-I. The bones were measured and growth expressed as percent increase in bone length over the 7-day culture period. RESULTS: When exposed to IL-1ß plus TNF-α (10 + 10 ng/ml), bone growth was markedly suppressed (6.6 ± 1.4 vs. 50.6 ± 2.5% in control bones; p < 0.001). The growth of cytokine exposed bones (IL-1ß plus TNF-α) was dose-dependently rescued by anakinra (0.05-500 µg/ml) or etanercept (0.5-500 µg/ml); at the highest concentrations, growth was similar as in control bones never exposed to cytokines. Also when combining IGF-I (100 ng/ml) and relatively low concentrations of anakinra (0.05 µg/ml) or etanercept (5 µg/ml), growth was rescued in an additive way. CONCLUSION: Etanercept and anakinra efficiently and dose-dependently prevent cytokine-induced bone growth impairment, and combination with IGF-I further improves bone growth.

Phosphate regulates embryonic endochondral bone development.

Phosphate is required for terminal differentiation of hypertrophic chondrocytes during postnatal growth plate maturation. In vitro models of chondrocyte differentiation demonstrate that 7 mM phosphate, a concentration analogous to that of the late gestational fetus, activates the mitochondrial apoptotic pathway in hypertrophic chondrocytes. This raises the question as to whether extracellular phosphate modulates chondrocyte differentiation and apoptosis during embryonic endochondral bone formation. To address this question, we performed investigations in the mouse metatarsal culture model that recapitulates in vivo bone development. Metatarsals were cultured for 4, 8, and 12 days with 1.25 and 7 mM phosphate. Metatarsals cultured with 7 mM phosphate showed a decrease in proliferation compared to those cultured in 1.25 mM phosphate. This decrease in proliferation was accompanied by an early enhancement in hypertrophic chondrocyte differentiation, associated with an increase in FGF18 expression. By 8 days in culture, an increase caspase-9 activation and apoptosis of hypertrophic chondrocytes was observed in the metatarsals cultured in 7 mM phosphate. Immunohistochemical analyses of embryonic bones demonstrated activation of caspase-9 in hypertrophic chondrocytes, associated with vascular invasion. Thus, these investigations demonstrate that phosphate promotes chondrocyte differentiation during embryonic development and implicate a physiological role for phosphate activation of the mitochondrial apoptotic pathway during embryonic endochondral bone formation.