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.

The syndrome of proximal femur, fibula, and midline metatarsal long bone deficiencies.

Human lower limb congenital long bone deficiencies cluster primarily at three distinct skeletal locations. Proximal femoral and fibular reductions are known phenomena. In contrast, midline metatarsal deficiencies have been misrepresented as lateral. The popular term, "fibular hemimelia," is inaccurate and its use is discouraged. All three locations correspond to discrete sites of evolving angiogenesis during transition from a single embryonic axial limb artery to the familiar and complex adult arterial pattern. Initiation of bone formation of cartilaginous primordia of the long bones at all three sites occurs in proximity to, and depends upon, successful invasion by mature nutrient vessels, formed during the 6th and 7th weeks of embryonic development. The adult arterial pattern is fully established by 8th embryonic week. Arterial transitions occur later in development, around the time of cessation of the molecular processes of patterning/specification of the embryonic limb. Evidence of flawed embryonic arterial transitions, involving missing, reduced and/or retained primitive vessels in association with congenital skeletal reductions have been demonstrated at all three sites. Current molecular models of limb development do not explain the distribution of this triad of congenital skeletal reductions. These dysmorphologies are most accurately described as post-specification errors of limb development. Recognition of this distinctive model of limb maldevelopment demands further investigation to create a more exact taxonomy, one consistent with both clinical and molecular criteria. The established terminologies originated by Frantz and O'Rahilly should be reconsidered or abandoned. Designation of this clinical triad as a syndrome of proximal femur, fibula, and midline metatarsal dystrophisms initiates that endeavor.

Greater Growth of Proximal Metatarsals in Bird Embryos and the Evolution of Hallux Position in the Grasping Foot.

In early theropod dinosaurs-the ancestors of birds-the hallux (digit 1) had an elevated position within the foot and had lost the proximal portion of its metatarsal. It no longer articulated with the ankle, but was attached at about mid-length of metatarsal 2 (mt2). In adult birds, the hallux is articulated closer to the distal end of mt2 at ground level with the other digits. However, on chick embryonic day 7, its position is as in early theropods at half-length of mt2. The adult distal location is acquired during embryonic days 8-10. To assess how the adult phenotype is acquired, we produced fate maps of the metatarsals of day 6 chicken embryos injecting the lipophilic tracer DiI. The fates of these marks indicate a larger expansion of the metatarsals at their proximal end, which creates the illusory effect that d1 moves distally. This larger proximal expansion occurs concomitantly with growth and early differentiation of cartilage. Histological analysis of metatarsals shows that the domains of flattened and prehypertrophic chondrocytes are larger toward the proximal end. The results suggest that the distal position of the hallux in the avian foot evolved as a consequence of an embryological period of expansion of the metatarsus toward the proximal end. It also brings attention to the developmental mechanisms leading to differential growth between epiphyses and their evolutionary consequences.

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.

MEPE is a novel regulator of growth plate cartilage mineralization.

Matrix extracellular phosphoglycoprotein (MEPE) belongs to the SIBLING protein family which play key roles in biomineralization. Although the growth plates of MEPE-overexpressing mice display severe morphological disruption, the expression and function of MEPE in growth plate matrix mineralization remains largely undefined. Here we show MEPE and its cleavage product, the acidic serine aspartate-rich MEPE-associated motif (ASARM) peptide, to be localised to the hypertrophic zone of the growth plate. We also demonstrate that the phosphorylated (p)ASARM peptide inhibits ATDC5 chondrocyte matrix mineralization. Stable MEPE-overexpressing ATDC5 cells also had significantly reduced matrix mineralization in comparison to the control cells. Interestingly, we show that the addition of the non-phosphorylated (np)ASARM peptide promoted mineralization in the ATDC5 cells. The peptides and the overexpression of MEPE did not affect the differentiation of the ATDC5 cells. For a more physiologically relevant model, we utilized the metatarsal organ culture model. We show the pASARM peptide to inhibit mineralization at two stages of development, as shown by histological and µCT analysis. Like in the ATDC5 cells, the peptides did not affect the differentiation of the metatarsals indicating that the effects seen on mineralization are direct, as is additionally confirmed by no change in alkaline phosphatase activity or mRNA expression. In the metatarsal organ cultures, the pASARM peptide also reduced endothelial cell markers and vascular endothelial growth factor mRNA expression. Taken together these results show MEPE to be an important regulator of growth plate chondrocyte matrix mineralization through its cleavage to an ASARM peptide.

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.

Identification of small molecular compounds and fabrication of its aqueous solution by laser-ablation, expanding primordial cartilage.

OBJECTIVE: The discovery of small molecular compounds that expand cartilage is needed. We searched for small molecular compounds that expand cartilage or enhance the actions of bone morphogenetic proteins (BMPs) on cartilage. DESIGN: Metatarsal primordial cartilage explants prepared from 14.5 days postcoitum (d.p.c.) mouse embryos were organ-cultured in the presence or absence of BMPs and/or 4-(5-Benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate (BPIB) and its related molecules. The perichondrium was removed from some of the cartilage explants by partial digestion with collagenase. BPIB aqueous solution was prepared by fragmenting BPIB crystals in water with laser irradiation and then added to cartilage explants in organ culture. RESULTS: We found that small molecular compounds, BPIB, available as SB431542 from Sigma and its related molecules, expand primordial cartilage explants in organ culture. These molecules are transforming growth factor-ß (TGF-ß) inhibitors, and the addition of excess TGF-ß reduced cartilage expansion induced by these molecules. The co-administration of BPIB and BMPs synergistically expanded cartilage explants. Removal of the perichondrium abolished BIPB-induced cartilage expansion but not BMP-induced cartilage-expansion, suggesting that BPIB, but not BMPs, expands cartilage through the perichondrium. Furthermore, we used the laser-ablation technique to generate BPIB aqueous solution in the presence of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) without the use of hazardous dimethyl sulfoxide (DMSO). The laser-ablation-generated BPIB aqueous solution was more stable, expanded cartilage explants more effectively than BPIB colloidal solution prepared with DMSO, and synergistically enhanced BMP-induced cartilage expansion. CONCLUSIONS: A small molecular compound, BPIB, expands primordial cartilage explants. A BPIB aqueous solution was created by laser-ablation without using DMSO and proved to be biologically active.

Catch-up growth after dexamethasone withdrawal occurs in cultured postnatal rat metatarsal bones.

Children exposed to systemic glucocorticoids often exhibit growth retardation and after the cessation of therapy catch-up growth occurs in many, but not all patients. The developmental regulation and underlying cellular mechanisms of catch-up growth are not fully understood. To clarify this issue, we established an in vitro model of catch-up growth. Here we present a protocol for the long-term culture (up to 160 days) of fetal (E20) as well as postnatal (P8) rat metatarsal bones which allowed us to characterize ex vivo the phenomenon of catch-up growth without any influence by systemic factors. The relevance of the model was confirmed by the demonstration that the growth of fetal and postnatal bones were stimulated by IGF1 (100 ng/ml) and inhibited by dexamethasone (Dexa; 1 microM). We found that the capacity to undergo catch-up growth was restricted to postnatal bones. Catch-up growth occurred after postnatal bones had been exposed to Dexa for 7 or 12 days but not after a more prolonged exposure (19 days). Incomplete catch-up growth resulted in compromised bone length when assessed at the end of the 4-month period of culture. While exposure to Dexa was associated with decreased chondrocyte proliferation and differentiation, catch-up growth was only associated with increased cell proliferation. We conclude that the phenomenon of catch-up growth after Dexa treatment is intrinsic to the growth plate and primarily mediated by an upregulation of chondrocyte proliferation.

Osteoclasts are important for bone angiogenesis.

Increased osteoclastogenesis and angiogenesis occur in physiologic and pathologic conditions. However, it is unclear if or how these processes are linked. To test the hypothesis that osteoclasts stimulate angiogenesis, we modulated osteoclast formation in fetal mouse metatarsal explants or in adult mice and determined the effect on angiogenesis. Suppression of osteoclast formation with osteoprotegerin dose-dependently inhibited angiogenesis and osteoclastogenesis in metatarsal explants. Conversely, treatment with parathyroid hormone related protein (PTHrP) increased explant angiogenesis, which was completely blocked by osteoprotegerin. Further, treatment of mice with receptor activator of nuclear factor-kappaB ligand (RANKL) or PTHrP in vivo increased calvarial vessel density and osteoclast number. We next determined whether matrix metalloproteinase-9 (MMP-9), an angiogenic factor predominantly produced by osteoclasts in bone, was important for osteoclast-stimulated angiogenesis. The pro-angiogenic effects of PTHrP or RANKL were absent in metatarsal explants or calvaria in vivo, respectively, from Mmp9(-/-) mice, demonstrating the importance of MMP-9 for osteoclast-stimulated angiogenesis. Lack of MMP-9 decreased osteoclast numbers and abrogated angiogenesis in response to PTHrP or RANKL in explants and in vivo but did not decrease osteoclast differentiation in vitro. Thus, MMP-9 modulates osteoclast-stimulated angiogenesis primarily by affecting osteoclasts, most probably by previously reported migratory effects on osteoclasts. These results clearly demonstrate that osteoclasts stimulate angiogenesis in vivo through MMP-9.

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.

Interference with cellular differentiation by D-serine through antagonism at N-methyl-D-aspartate receptors composed of NR1 and NR3A subunits in chondrocytes.

Serine racemase (SR) is responsible for the biosynthesis of D-serine (D-Ser), an endogenous co-agonist for the glycine (Gly)-binding site on N-methyl-D-aspartate (NMDA) receptors, from L-Ser in the brain. We have previously demonstrated high expression of SR by chondrocytes in cartilage. In this study, we attempted to elucidate the possible functional role of D-Ser in chondrogenesis. Expression of mRNA and corresponding protein was seen for SR in cultured rat costal chondrocytes, while the addition of L-Ser significantly increased intracellular and extracellular levels of D-Ser. In organotypic cultured mouse embryonic metatarsals isolated before vascularization, SR mRNA was highly localized in hypertrophic and calcified chondrocytes. Exposure to D-Ser not only suppressed several chondrocytic maturation markers, including alkaline phosphatase (ALP) activity, Ca2+ accumulation, nodule formation, and osteopontin expression, in rat chondrocytes, but also delayed chondral mineralization in mouse metatarsals. Either NMDA or Gly alone significantly increased Ca2+ accumulation in cultured chondrocytes, whereas D-Ser significantly prevented Ca2+ accumulation by Gly, but not by NMDA. Gly alone also significantly increased gene transactivation by the introduction of runt-related transcription factor-2 (Runx2) in COS7 cells transfected with NR1 and NR3A subunits, while D-Ser significantly prevented the increase by Gly without affecting the promoter activity of Runx2. In both cultured chondrocytes and metatarsals from NR1-null mice, significant decreases were seen in ALP activity and chondral mineralization, respectively. These results suggest that D-Ser may negatively regulate cellular differentiation through inhibiting NMDA receptors composed of NR1 and NR3A subunits in a manner related to Runx2 transcriptional activity in chondrocytes.

Androgen receptor modulation does not affect longitudinal growth of cultured fetal rat metatarsal bones.

BACKGROUND: Systemic administration of the nonaromatizable androgen oxandrolone stimulates growth in girls with Turner syndrome and boys with a constitutional delay of growth and puberty. It is unknown if oxandrolone acts locally at the growth plate level to stimulate longitudinal bone growth. METHODS: Metatarsal bones from female and male rat fetuses (day E20) were cultured for 14 days in the presence of oxandrolone, testosterone or the androgen receptor (AR) antagonist flutamide with/without insulin-like growth-factor-I (IGF-I) or charcoal-treated serum. RESULTS: The AR was found to be expressed in both male and female fetal rat metatarsal bones. Neither oxandrolone nor testosterone had any effect on metatarsal bone growth when tested at a wide concentration range (1 nM to 10 microM), not even in the presence of IGF-I (100 ng/ml) or charcoal-treated serum (10%). Bone growth was also unaffected when the AR was blocked by flutamide. Control experiments confirmed that metatarsal bone growth was significantly stimulated by IGF-I (p < 0.001). CONCLUSION: Modulation of AR activity in the fetal rat growth plate does not affect linear bone growth. Extrapolating from these in vitro data, it could be speculated that oxandrolone stimulates longitudinal bone growth in treated children by acting indirectly rather than directly through AR activation in growth plate chondrocytes.

Does in vitro low-intensity pulsed ultrasound stimulate endochondral ossification?

The objective of this study was to examine the effects of low-intensity pulsed ultrasound treatment of murine fetal metatarsal (MT) bone anlagen in vitro. Metatarsal preparations of 17 mice fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days with and without low-intensity ultrasound stimulation. The total length of the metatarsal rudiments and the length of the calcified diaphysis were measured at days 1, 3, 5, and 7. After 7 days in culture, histological and histomorphometric analyses were performed. The increase in total length of the metatarsal bones and in length of the calcified diaphysis during in vitro culture was not affected by ultrasound treatment. Histological analysis of the MT preparations after 7 days of in vitro culture showed a healthy appearance of all specimens and revealed no differences in the general histological outcome between the stimulated and control groups. All histomorphometric parameters were unaffected by ultrasound stimulation, except for the length of the proximal hypertrophic zone which was significantly shorter in the stimulated bones compared to controls (p=0.043). Our results illustrate no stimulating effect of ultrasound treatment on endochondral ossification which may be based on different experimental conditions in comparison to other studies demonstrating a positive effect of sonication. Thus, ultrasonically induced stimulatory effects on endochondral ossification seem to be highly dependent on experimental conditions.

IGF-I signalling in bone growth: inhibitory actions of dexamethasone and IL-1beta.

OBJECTIVE: To determine if glucocorticoids and proinflammatory cytokines inhibit bone growth through a common mechanism involving impaired IGF-I signalling. DESIGN: IGF-I (100 ng/ml), dexamethasone (dex) (10(-6)M) and IL-1beta (10 ng/ml) with inhibitors of the PI3K (LY294002) and Erk 1/2 (PD98059 and UO126) IGF-I pathways (all 10 microM) were studied using the ATDC5 chondrocyte cell line and murine fetal metatarsal cultures. RESULTS: IGF-I stimulated ATDC5 chondrocyte proliferation (322%; P < 0.001 versus control). Addition of PD or LY individually to IGF-I supplemented ATDC5 cultures partially reduced proliferation by 32% (P < 0.001), and 66% (P < 0.001), respectively. PD and LY in combination blocked all IGF-I stimulated ATDC5 proliferation. LY significantly reversed IGF-I stimulatory effects on metatarsal growth (P < 0.001), whereas PD and UO treatment had no effect. IGF-I induced ATDC5 proliferation was further decreased when Dex (24%; P < 0.01) or IL-1beta (33%; P < 0.001) were added to PD but not LY cultures. Metatarsal growth inhibition by LY was unaltered by Dex or IL-1beta addition. CONCLUSIONS: Both the PI3K and Erk 1/2 pathways contributed independently to IGF-I mediated ATDC5 proliferation. However in metatarsal cultures, the Erk 1/2 pathway was not required for IGF-I stimulated growth. Dex and IL-1beta may primarily inhibit IGF-I induced bone growth through the PI3K pathway.

X-ray evaluation of symmetry development of human metatarsal bones in different periods of fetal life.

BACKGROUND: Symmetry and asymmetry have intrigued people for centuries. Most studies are based on adult specimens, and there are few asymmetry studies carried out on children and fetuses. The aim of this study was an x-ray evaluation of the asymmetry of human metatarsal bones in different periods of fetal life. MATERIAL/METHODS: The material consisted of 36 fetuses (18 male, 18 female). The fetuses were preserved in 10% formalin and the metatarsal bones were subsequently x-rayed in the A-P projection. Total area and length of the metatarsal bones were measured. The fetuses were divided into three groups according to fetal age calculated by measurement of the crown-rump length (CRL) and total length using the Scammon and Calkins methods. The results were put into tables which show which side of a sample bone pair is larger. RESULTS: Structural asymmetry in fetal metatarsal bones on the right and left side was found. In the younger group, the character of the asymmetry fluctuated. A clear one-sided domination in all metatarsal bones appeared only in the oldest group (over 26 gestational weeks). CONCLUSIONS: These result demonstrate that such asymmetry forms and matures during ontogenesis, similarly to other systems and organs.

Endochondral ossification in vitro is influenced by mechanical bending.

Bone development is influenced by the local mechanical environment. Experimental evidence suggests that altered loading can change cell proliferation and differentiation in chondro- and osteogenesis during endochondral ossification. This study investigated the effects of three-point bending of murine fetal metatarsal bone anlagen in vitro on cartilage differentiation, matrix mineralization and bone collar formation. This is of special interest because endochondral ossification is also an important process in bone healing and regeneration. Metatarsal preparations of 15 mouse fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days. After 3 days in culture to allow adherence they were stimulated 4 days for 20 min twice daily by a controlled bending of approximately 1000-1500 microstrain at 1 Hz. The paraffin-embedded bone sections were analyzed using histological and histomorphometrical techniques. The stimulated group showed an elongated periosteal bone collar while the total bone length was not different from controls. The region of interest (ROI), comprising the two hypertrophic zones and the intermediate calcifying diaphyseal zone, was greater in the stimulated group. The mineralized fraction of the ROI was smaller in the stimulated group, while the absolute amount of mineralized area was not different. These results demonstrate that a new device developed to apply three-point bending to a mouse metatarsal bone culture model caused an elongation of the periosteal bone collar, but did not lead to a modification in cartilage differentiation and matrix mineralization. The results corroborate the influence of biophysical stimulation during endochondral bone development in vitro. Further experiments with an altered loading regime may lead to more pronounced effects on the process of endochondral ossification and may provide further insights into the underlying mechanisms of mechanoregulation which also play a role in bone regeneration.

Novel late response genes of PTHrP in chondrocytes.

To gain more insight into the downstream effectors of parathyroid hormone (PTH) related peptide (PTHrP) signaling in chondrocytes, we performed microarray analysis to identify late PTHrP response genes using the chondrogenic ATDC5 cell line and studied their response in the osteoblastic KS483 cell line and explanted metatarsals. At day 8 of micromass culture, ATDC5 cells have pre-hypertrophic-like characteristics and at this time point the cells were stimulated with PTHrP for 24 and 72 h and RNA was isolated. PTHrP treatment inhibited outgrowth of cartilage matrix and decreased the expression of Col10a1 mRNA, which is in line with the inhibitory effects of PTHrP on chondrocyte differentiation. Using cDNA microarray analysis, a list of 9 genes (p< 10(-3)) was generated, including 3 upregulated (IGFBP4, Csrp2, and Ecm1) and 6 downregulated (Col9a1, Col2a1, Agc, Hmgn2, Calm1, and Mxd4) response genes. Four out of 9 genes are novel PTHrP response genes and 2 out of 9 have not yet been identified in cartilage. Four out of 9 genes are components of the extra-cellular matrix and the remaining genes are involved in signal transduction and transcription regulation. The response to PTHrP was validated by quantitative PCR, using the same RNA samples as labeled in the microarray experiments and RNA samples isolated from a new experiment. In addition, we examined whether these genes also reacted to PTHrP in other PTHrP responsive models, like KS483 osteoblasts and explanted metatarsals. The expression of late PTHrP response genes varied between ATDC5 chondrocytes, KS483 osteoblasts and metatarsals, suggesting that the expression of late response genes is dependent on the cellular context of the PTHrP responsive cells.

Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent.

Proinflammatory cytokines inhibit growth plate development. However, their underlying mechanisms of action are unclear. These effects may be mediated by ceramide, a sphingosine-based lipid second messenger, which is elevated in a number of chronic inflammatory diseases. To test this hypothesis, we determined the effects of C2-ceramide, a cell permeable ceramide analogue, on the growth of the ATDC5 chondrogenic cell line and on cultured fetal mice metatarsals. In ATDC5 cells, C2-ceramide significantly induced apoptosis at both 40 (82%; P < 0.05) and 25 microM (53%; P < 0.05). At 40 microM, C2-ceramide significantly reduced proliferation ([3H]-thymidine uptake/mg protein) (62%; P < 0.05). C2-ceramide did not markedly alter the differentiation state of the cells as judged by the expression of markers of chondrogenesis and differentiation (sox 9, collagen II and collagen X). The IGF-I signalling pathway is the major autocrine/paracrine regulator of bone growth. Both in the presence and absence of IGF-I, C2-ceramide (25 microM) induced an equivalent reduction in proliferation (60%; P < 0.001). Similarly, C2-ceramide (40 microM) induced a 31% reduction in fetal metatarsal growth both in the presence and absence of IGF-I (both P < 0.001). Furthermore, C2-ceramide reduced ADCT5 proliferation in the presence of AG1024, an IGF-I and insulin receptor blocker. Therefore, C2-ceramide-dependent inhibition appears to be independent of IGF-mediated stimulation of bone growth. Indeed, biochemical studies demonstrated that C2-ceramide (25 microM) pretreatment did not alter IGF-I-stimulated phosphorylation of insulin receptor substrate-1, Akt or P44/42 MAP kinase. In conclusion, C2-ceramide inhibits proliferation and induces apoptosis in growth plate chondrocytes through an IGF-I independent mechanism.