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Background: Discoid meniscus (DM) is a rare variant of regular knee anatomy. Compared to standard meniscus it is thicker and abnormal in shape; these characteristics make it more prone to tear. It is a congenital defect whose correct etiology is still debated and far from being clarified. The purpose of this systematic review is to evaluate evidences of DM in human fetuses in order to assess whether embryological development may have a role., Methods: A systematic review was performed on PubMed, Scopus, and Embase with different combinations of the keywords "discoid meniscus", "embryology", "fetus", "neonatal". Search yielded 1013 studies, on which we performed a primary evaluation., Results: Seven studies were considered including a total of 1378 fetal menisci specimens, from 396 different fetuses. Discoid shape was not found represented as a normal stage of prenatal development. From 782 lateral menisci analyzed, only 86 (10.86%) were discoid (13 complete, 73 incomplete type). None of medial menisci was found to be discoid. Lateral meniscus was observed to cover a larger surface of tibial plateau than medial one until 28th gestational week., Conclusion: Lateral meniscus seems to be more prone to discoid shape for its natural tendency of covering a larger surface of the tibial plateau during fetal stages. However the fact that a discoid shape was not found in the majority of fetuses suggests that it is not a normal stage of fetal development. To support a single etiological factor it will be appropriate to have further morphological and morphometric studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: The present study established a nomogram of fetal thyroid circumference (FTC) and the appearance timing of fetal distal femoral and proximal tibial ossification to assess fetal thyroid function in Japan., Methods: Between April 2015 and July 2019, normal pregnant women at our hospital were recruited for the study. FTC was measured by the automatic ellipse outline and plotted against gestational age (GA). Fetal distal femoral and proximal tibial ossification measurements were obtained with standard electronic calipers from outer-to-outer margins (> 1 mm as the presence of ossification)., Results: A total of 199 pregnant women were examined. FTC increased logarithmically to GA. A nomogram of FTC was expressed by a logarithmic formula: [Formula: see text]. The respective 5-95th percentiles of FTC at each GA were 20.2-36.2 mm at 22 weeks, 25.0-44.8 mm at 26 weeks, 29.2-52.3 mm at 30 weeks, and 32.9-59.0 mm at 34 weeks. The fetal distal femoral epiphysis was not visualized before 30 weeks, but was visualized in 100% of fetuses after 35 weeks of gestation. The fetal proximal tibial epiphysis was not visualized before 33 weeks, but was visualized in 73.7% of fetuses at 37 weeks of gestation., Conclusion: We generated a GA-dependent FTC nomogram for Japanese fetuses. We also confirmed the appearance timing of fetal distal femoral and proximal tibial ossification to assess bone maturation. These assessments may be very useful for evaluating fetal thyroid function in Japan.

The vertebrate limb serves as an experimental paradigm to study mechanisms that regulate development of the stereotypical skeletal elements. In this study, we simultaneously inactivated Sall4 using Hoxb6Cre and Plzf in mouse embryos, and found that their combined function regulates development of the proximal-anterior skeletal elements in hindlimbs. The Sall4 ; Plzf double knockout exhibits severe defects in the femur, tibia, and anterior digits, distinct defects compared to other allelic series of Sall4 ; Plzf We found that Sall4 regulates Plzf expression prior to hindlimb outgrowth. Further expression analysis indicated that Hox10 genes and GLI3 are severely downregulated in the Sall4 ; Plzf double knockout hindlimb bud. In contrast, PLZF expression is reduced but detectable in Sall4 ; Gli3 double knockout limb buds, and SALL4 is expressed in the Plzf ; Gli3 double knockout limb buds. These results indicate that Plzf , Gli3 , and Hox10 genes downstream of Sall4 , regulate femur and tibia development. In the autopod, we show that Sall4 negatively regulates Hedgehog signaling, which allows for development of the most anterior digit. Collectively, our study illustrates genetic systems that regulate development of the proximal-anterior skeletal elements in hindlimbs., (Copyright © 2020 by the Genetics Society of America.)

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy human bones makes particularly valuable the use of mammalian models, such as mouse and rat, to look into different aspects of bone growth and homeostasis. Additionally, the development of sophisticated genetic tools in mice allows more complex studies of long bone growth and asks for an expansion of techniques used to study bone growth. Here, we present a detailed protocol for ex vivo murine bone culture, which allows the study of bone and cartilage in a tightly controlled manner while recapitulating most of the in vivo process. The method described allows the culture of a range of bones, including tibia, femur, and metatarsal bones, but we have focused mainly on tibial culture here. Moreover, it can be used in combination with other techniques, such as time-lapse live imaging or drug treatment.

Joint morphogenesis is the process during which distinct and functional joint shapes emerge during pre- and post-natal joint development. In this study, a repeatable semi-automatic protocol capable of providing a 3D realistic developmental map of the prenatal mouse knee joint was designed by combining Optical Projection Tomography imaging (OPT) and a deformable registration algorithm (Sheffield Image Registration toolkit, ShIRT). Eleven left limbs of healthy murine embryos were scanned with OPT (voxel size: 14.63μm) at two different stages of development: Theiler stage (TS) 23 (approximately 14.5 embryonic days) and 24 (approximately 15.5 embryonic days). One TS23 limb was used to evaluate the precision of the displacement predictions for this specific case. The remaining limbs were then used to estimate Developmental Tibia and Femur Maps. Acceptable uncertainties of the displacement predictions computed from repeated images were found for both epiphyses (between 1.3μm and 1.4μm for the proximal tibia and between 0.7μm and 1.0μm for the femur, along all directions). The protocol was found to be reproducible with maximum Modified Housdorff Distance (MHD) differences equal to 1.9 μm and 1.5 μm for the tibial and femoral epiphyses respectively. The effect of the initial shape of the rudiment affected the developmental maps with MHD of 21.7 μm and 21.9 μm for the tibial and femoral epiphyses respectively, which correspond to 1.4 and 1.5 times the voxel size. To conclude, this study proposes a repeatable semi-automatic protocol capable of providing mean 3D realistic developmental map of a developing rudiment allowing researchers to study how growth and adaptation are directed by biological and mechanobiological factors., Competing Interests: The authors of this manuscript have no affiliation with or involvement in any organization with any financial interest in the subject matter or material discussed in this manuscript. The Certara QSP, Certara UK Limited affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

Purposes: Tibial shaft ossification in terms of its size and growth may be criticalin describing both the fetal stage and maturity, and in identifying innate disorders. The present study was executed to quantitatively assess ossification of the tibial shaft, taking its morphometric linear, planar and volumetric parameters into account., Materials and Methods: With the use of methods of CT, digital-image analysis and statistics, the evolutionof tibial shaft ossification in 47 spontaneously aborted human fetuses at the age of 17-30 weeks was studied., Results: Without any male-female and right-left morphometric differences, the best fit growth dynamics fortibial shaft ossification was modelled by the following functions: y = 5.312 + 0.034 × (age) 2  ± 0.001 (R 2  = 0.89) for its length, y = - 2.855 + 0.307 × age ± 0.009 (R 2  = 0.96) for its proximal transverse diameter, y = - 0.758 + 0.153 × age ± 0.005 (R 2  = 0.88) for its middle transverse diameter, y = - 1.844 + 0.272 × age ± 0.09 (R 2  = 0.90) for its distal transverse diameter, y = - 40.263 + 0.258 × (age) 2  ± 0.007 (R 2  = 0.94) for its projection surface area, and y = - 287.996 + 1.186 × (age) 2  ± 0.037 (R 2  = 0.92) for its volume. The femoral-to-tibial ossification length ratio was 1.15 ± 0.1., Conclusions: The size of tibial shaft ossification displays neither sex nor laterality differences. Tibial shaft ossification follows quadratic functions with respect to its length, projection surface area and volume, and linear functions with respect to its proximal, middle and distal transverse diameters. The obtained morphometric data of tibial shaft ossification are considered normative age-specific references of relevance in both the estimation of fetal ages and the ultrasound diagnostics of congenital defects.

This study investigated the mechanisms underlying the retarded development of long bone in fetus by prenatal nicotine exposure (PNE) which had been demonstrated by our previous work. Nicotine (2.0 mg/kg.d) or saline was injected subcutaneously into pregnant rats every morning from gestational day (GD) 9 to 20. Fetal femurs or tibias were harvested for analysis on GD 20. We found massive accumulation of hypertrophic chondrocytes and a delayed formation of primary ossification center (POC) in the fetal femur or tibia of rat fetus after PNE, which was accompanied by a decreased amount of osteoclasts in the POC and up-regulated expression of osteoprotegerin (OPG) but by no obvious change in the expression of receptor activator of NF-κB ligand (RANKL). In primary osteoblastic cells, both nicotine (0, 162, 1620, 16,200 ng/ml) and corticosterone (0, 50, 250, 1250 nM) promoted the mRNA expression of OPG but concentration-dependently suppressed that of RANKL. Furthermore, blocking α4β2-nicotinic acetylcholine receptor (α4β2-nAChR) or glucocorticoid receptor rescued the above effects of nicotine and corticosterone, respectively. In conclusion, retarded osteoclastogenesis may contribute to delayed endochondral ossification in long bone in fetal rats with PNE. The adverse effects of PNE may be mediated via the direct effect of nicotine and indirect effect of maternal corticosterone on osteoblastic cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Since healing of anterior cruciate ligament (ACL) grafts occurs by formation of a fibrovascular scar-tissue interface rather than by reformation of the native fibrocartilage transition zone, the purpose of our study was to examine expression of various signaling molecules and transcription factors that are known to be involved in embryologic insertion-site development following ACL reconstruction. We also aimed to characterize a murine model of ACL reconstruction to allow future study of the molecular mechanisms of healing., Methods: Seventy-nine mice underwent reconstruction of the ACL with autograft. Healing was assessed using histology in 12 mice and quantitative real-time polymerase chain reaction (qRT-PCR) gene-expression analysis in 3 mice at 1 week postoperatively (Group-1 mice) and by biomechanical analysis in 7, histological analysis in 7, immunohistochemical analysis in 5, microcomputed tomography analysis in 5, and qRT-PCR analyses in 8 at 2 weeks (Group-2 mice) and 4 weeks (Group-3 mice) postoperatively. Fifteen additional mice did not undergo surgery and were used for biomechanical (7 mice), qRT-PCR (3 mice), and immunohistochemical (5 mice) analyses to obtain baseline data for the native ACL., Results: Histological analysis demonstrated healing by formation of fibrovascular tissue at the tendon-bone interface. Immunohistochemical analysis showed a positive expression of proteins in the Indian hedgehog, Wnt, and parathyroid hormone-related protein (PTHrP) pathways. There was minimal Sox-9 expression. Gene-expression analysis showed an initial increase in markers of tissue repair and turnover, followed by a subsequent decline. Mean failure force and stiffness of the native ACL were 5.60 N and 3.44 N/mm, respectively. Mean failure force and stiffness were 1.29 N and 2.28 N/mm, respectively, in Group 2 and were 1.79 N and 2.59 N/mm, respectively, in Group 3, with 12 of 14 failures in these study groups occurring by tunnel pull-out., Conclusions: The spatial and temporal pattern of expression of signaling molecules that direct embryologic insertion-site formation was not adequate to restore the structure and composition of the native insertion site., Clinical Relevance: Development of a murine model to study ACL reconstruction will allow the use of transgenic animals to investigate the cellular, molecular, and biomechanical aspects of tendon-to-bone healing following ACL reconstruction, ultimately suggesting methods to improve healing in patients.

Some epiphyseal growth cartilage canals are surrounded by a ring of hypereosinophilic matrix consisting of collagen type I. Absence of the collagen type I ring may predispose canal vessels to failure and osteochondrosis, which can lead to fragments in joints (osteochondrosis dissecans). It is not known whether the ring develops in response to programming or biomechanical force. The distribution that may reveal the function of the ring has only been described in the distal femur of a limited number of foals. It is also not known which cells are responsible for producing the collagen ring. The aims of the current study were to examine fetuses and foals to infer whether the ring forms in response to biomechanical force or programming, to describe distribution and to investigate which cell type produces the ring. The material consisted of 46 fetuses and foals from 293 days of gestation to 142 days old, of both sexes and different breeds, divided into three groups, designated the naïve group up to and including the day of birth, the adapting group from 2 days up to and including 14 days old, and the loaded group from 15 days and older. The distal tibia was sawn into parasagittal slabs and the cranial half of the central slab from the intermediate ridge was examined by light microscopy and immunohistochemical staining for collagen type I. Presence, completeness and location of the collagen ring was compared, as was the quantity of perivascular mesenchymal cells. An eosinophilic ring present on HE-stained sections was seen in every single fetus and foal examined, which corresponded to collagen type I in immunostained sections. A higher proportion of cartilage canals were surrounded by an eosinophilic ring in the naïve and adapting groups at 73 and 76%, respectively, compared with the loaded group at 51%. When considering only patent canals, the proportion of canals with an eosinophilic ring was higher in the adapting and loaded than the naïve group of foals. The ring was present around 90 and 81% of patent canals in the deep and middle layers, respectively, compared with 58% in the superficial layer, and the ring was more often complete around deep compared with superficial canals. The ring was absent or partial around chondrifying canals. When an eosinophilic ring was present around patent canals, it was more common for the canal to contain one or more layers of perivascular mesenchymal cells rather than few to no layers. It was also more common for the collagen ring to be more complete around canals that contained many as opposed to few mesenchymal cells. In conclusion, the proportion of cartilage canals that had an eosinophilic ring was similar in all three groups of fetuses and foals, indicating that the presence of the collagen ring was mostly programmed, although some adaptation was evident. The ring was more often present around deep, compared with superficial canals, indicating a role in preparation for ossification. The collagen ring appeared to be produced by perivascular mesenchymal cells., (© 2017 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Autosomal-recessive omodysplasia (OMOD1) is a genetic condition characterized by short stature, shortened limbs, and facial dysmorphism. OMOD1 is caused by loss-of-function mutations of glypican 6 ( GPC6 ). In this study, we show that GPC6 -null embryos display most of the abnormalities found in OMOD1 patients and that Hedgehog (Hh) signaling is significantly reduced in the long bones of these embryos. The Hh-stimulatory activity of GPC6 was also observed in cultured cells, where this GPC increased the binding of Hh to Patched 1 (Ptc1). Consistent with this, GPC6 interacts with Hh through its core protein and with Ptc1 through its glycosaminoglycan chains. Hh signaling is triggered at the primary cilium. In the absence of Hh, we observed that GPC6 is localized outside of the cilium but moves into the cilium upon the addition of Hh. We conclude that GPC6 stimulates Hh signaling by binding to Hh and Ptc1 at the cilium and increasing the interaction of the receptor and ligand., (© 2017 Capurro et al.)

Objective: Statins are widely used drugs for cholesterol lowering, which were recently found to counteract the effects of aberrant fibroblast growth factor receptor (FGFR3) signaling in cell and animal models of FGFR3-related chondrodysplasia. This opened an intriguing therapeutic possibility for human dwarfing conditions caused by gain-of-function mutations in FGFR3, although the mechanism of statin action on FGFR3 remains unclear. Here, we determine the effect of statins on FGFR signaling in chondrocytes., Design: Cultured chondrocyte cell lines, mouse embryonic tibia cultures and limb bud micromasses were treated with FGF2 to activate FGFR signaling. The effects of atorvastatin, fluvastatin, lovastatin and pravastatin on FGFR3 protein stability and on FGFR-mediated chondrocyte growth-arrest, loss of extracellular matrix (ECM), induction of premature senescence and hypertrophic differentiation were evaluated., Results: Statins did not alter the level of FGFR3 protein expression nor produce any effect on FGFR-mediated inhibition of chondrocyte proliferation and hypertrophic differentiation in cultured chondrocyte cell lines, mouse tibia cultures or limb bud micromasses., Conclusion: We conclude that statins do not inhibit the FGFR signaling in chondrocytes. Therefore the statin-mediated rescue of FGFR3-related chondrodysplasia, described before, is likely not intrinsic to the growth plate cartilage., (Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta-like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast-specific manner, we investigated the ligand-specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1-induced Notch signaling was responsible for the expansion of the bone-forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1-Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast-specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast-osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1-mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand-specific activation of Notch signaling in the maintenance of bone homeostasis. J. Cell. Physiol. 232: 2569-2580, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc., (© 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.)

Tibial hemimelia is a rare lower-extremity pre-axial longitudinal deficiency characterized by complete or partial absence of the tibia. The reported incidence is 1 in 1 million live births. In this pictorial essay, we define tibial hemimelia and describe associated conditions and principles of preoperative imaging assessment for a child with tibial hemimelia. We also indicate the imaging findings that might influence the choice of treatment, describe the most widely used classification systems, and briefly discuss current treatment approaches.

The neuropeptide Calcitonin Gene-Related Peptide (CGRP) is a well-characterized neurotransmitter. However, little is known about the role of CGRP in osteogenesis and vascular genesis during the developmental formation of bone. In the present study, we assessed the abundance of CGRP mRNA and the mRNA of osteogenesis and vascular genesis markers in the foetal mouse mandible and leg bone (tibia). We also analysed the expression and localization of CGRP, osteopontin (OPN) and vascular endothelial growth factor (VEGF-A) using in situ hybridization and immunohistochemical localization in the mouse mandible and tibia at embryonic days 12.5 (E12.5), E14.5, E17.5, and postnatal day 1 (P1). CGRP was clearly detected in the mandible relative to the tibia at E14.5. Hybridization using an anti-sense probe for CGRP was not detected in the mandible at P1. Hybridization with an anti-sense probe for OPN was detected at E14.5, later in the mandible and at P1 in Meckel's cartilage. However, OPN was only detected in the tibia at E17.5 and later. The abundance of CGRP mRNA differed between the mandible and tibia. The level of vasculogenesis markers, such as VEGF-A, was similar to that of CGRP in the mandible. The levels of VEGF-A, cluster of differentiation 31 (CD31) and lymphatic vessel endothelial hyaluronan receptor 1 (LIVE-1) differed from that of OPN in the mandible. In contrast, the levels of VEGF-A, CD31, matrix metalloproteinase-2 (MMP-2), collagen I (Col I), collagen II (Col II) and OPN mRNA differed from E12.5 to P1 (P<0.001) in the tibia. The abundance of mRNA of CGRP and bone matrix markers (Col I, Col II, and OPN) was low at P5 in the tibia. These differences in CGRP and other mRNAs may induce a different manner of ossification between the mandible and tibia. Therefore, a time lag of ossification occurs between the mandible and tibia during foetal development.

The effects of experimentally induced high incubation temperature on the embryonic development of growth plate of the chicken were investigated by means of histological and enzyme histochemical methods. In the experiments, 250 fertile eggs of Ross-308 broiler strain were divided into two groups, the control eggs were maintained under optimal conditions (37.8°C and 65% ± 2% relative humidity, rh) during the whole incubation period. Heat-stress imposed eggs were maintained under normal conditions (37.8°C and 65% ± 2% rh) until the 10th day of incubation, and then, continuously (24 h per day) exposed to high temperature (38.8°C and 65% ± 2% rh). Tissue samples were taken from 10 animals of each group at the 11th, 13th, 15th, 18th, 21st days of incubation. Tissue samples were processed by enzyme histochemical methods in addition to routine histological techniques. The relative tibia weights and tibia length were lower in the heat-stress group compared to the control group. The results of the measurements of the growth plate showed that the proliferative zone was narrowed whereas, the transitional and hypertrophic zone were thickened in the heat stress group. Alkaline phosphatase (ALP) density was significantly decreased in the heat-stress group compared to the control group. In conclusion, bone formation and growth plate formation are crucial for embryo development and 1°C higher from optimum may increase the incidence of skeletal disorders and leg problems in broiler chickens which is one of the major animal welfare concerns for the poultry industry., (© 2016 Wiley Periodicals, Inc.)

This study aimed to analyze the spatial developmental changes of rat cruciate ligaments by three-dimensional (3D) reconstruction using episcopic fluorescence image capture (EFIC). Cruciate ligaments of Wister rat embryos between embryonic day (E) 16 and E20 were analyzed. Samples were sectioned and visualized using EFIC. 3D reconstructions were generated using Amira software. The length of the cruciate ligaments, distances between attachment points to femur and tibia, angles of the cruciate ligaments and the cross angle of the cruciate ligaments were measured. The shape of cruciate ligaments was clearly visible at E17. The lengths of the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) increased gradually from E17 to E19 and drastically at E20. Distances between attachment points to the femur and tibia gradually increased. The ACL angle and PCL angle gradually decreased. The cross angle of the cruciate ligaments changed in three planes. The primordium of the 3D structure of rat cruciate ligaments was constructed from the early stage, with the completion of the development of the structures occurring just before birth.

Background: Bone substation grafts, such as hydroxyapatite (HA) and tricalciumphosphate (TCP), have been extensively used in clinical applications, but evidence suggests that they offer poor osteoinductive properties compared to allografts and autografts. In order to increase bone growth with such grafts, Bone Morphogenetic Protein 2 (BMP-2) was incorporated into a three dimensional reservoir. The purpose of the present study was to develop a novel drug delivery system which is capable of controlled release of BMP-2., Methods: DBB were prepared from bovine cancellous bone harvested from fetal bovine femur or tibia and then sinting at 1000°C. BMP-2-loaded chitosan (CS) microspheres were fabricated by cross-linking. Then the treated DBB powders were blended with chitosan microspheres solution. Finally, the composites were lyophilized with a freeze dryer to obtain the DBB/CMs scaffolds. X-ray diffractor (XRD), scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) were used to characterize the sample. The quantification of the delivery profile of BMP-2 was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The in vitro assays were to characterize the biocompatibility of this composite., Results: In this study, BMP-2/Chitosan (CS) microspheres were successively loaded onto a deproteinized bovine bone (DBB) scaffold. The release profile of BMP-2 indicated an initial burst release followed by a more even sustained release. An in vitro bioactivity assay revealed that the encapsulated growth factor was biologically active., Conclusions: The cell culture assay suggest that the excellent biocompatibility of the DBB- BMP-2/CS. Therefore, this novel microsphere scaffold system can be effectively used in current tissue engineering applications.

Leg problems in broiler chickens may partly be prevented by providing optimal circumstances for skeletal development during incubation. One of the factors demonstrated to affect bone development is eggshell temperature (EST), which provides a reliable reflection of embryo temperature. The present experiment aimed to investigate the effect of EST on development and asymmetry of the femur, tibia, and metatarsus in broiler chicken hatchlings. Eggs were incubated from d 0 until hatch at 1 of 4 EST: low (36.9°C), normal (37.8°C), high (38.6°C), and very high (39.4°C). At hatch, chick quality was determined in terms of chick length, yolk-free body mass, navel score, and organ weights. Tibia, femur, and metatarsus were weighed, their length and width (mediolateral diameter) and depth (craniocaudal diameter) at the middle of the shaft were measured, and their ash content was determined. Relative asymmetry of the leg bones was determined from their relative dimensions. Hatchability, chick quality, and organ development were lower for very high EST compared with all other treatments. Very high EST resulted in lowest tibia and metatarsus lengths (-3.1 to -8.4%) compared with all other treatments, and lower metatarsus weight (-9.1%) and femur length (-4.9%) compared with high EST. Relative asymmetry and ash content did not differ among treatments and no relation between EST and bone parameters was found. To conclude, very high EST resulted in lower bone development, hatchability, and chick quality. Few differences in bone development and chick quality were found between low, normal, and high EST., (©2014 Poultry Science Association Inc.)

Objective: Development of the knee joint was morphologically investigated, and the process of cavitation was analyzed by using episcopic fluorescence image capture (EFIC) to create spatial and temporal three-dimensional (3D) reconstructions., Methods: Knee joints of Wister rat embryos between embryonic day (E)14 and E20 were investigated. Samples were sectioned and visualized using an EFIC. Then, two-dimensional image stacks were reconstructed using OsiriX software, and 3D reconstructions were generated using Amira software., Results: Cavitations of the knee joint were constructed from five divided portions. Cavity formation initiated at multiple sites at E17; among them, the femoropatellar cavity (FPC) was the first. Cavitations of the medial side preceded those of the lateral side. Each cavity connected at E20 when cavitations around the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) were completed., Conclusion: Cavity formation initiated from six portions. In each portion, development proceeded asymmetrically. These results concerning anatomical development of the knee joint using EFIC contribute to a better understanding of the structural feature of the knee joint., (Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Limb skeletal pattern relies heavily on graded Sonic hedgehog (Shh) signaling. As a morphogen and growth cue, Shh regulates identities of posterior limb elements, including the ulna/fibula and digits 2 through 5. In contrast, proximal and anterior structures, including the humerus/femur, radius/tibia, and digit 1, are regarded as Shh independent, and mechanisms governing their specification are unclear. Here, we show that patterning of the proximal and anterior limb skeleton involves two phases. Irx3 and Irx5 (Irx3/5) are essential in the initiating limb bud to specify progenitors of the femur, tibia, and digit 1. However, these skeletal elements can be restored in Irx3/5 null mice when Shh signaling is diminished, indicating that Shh negatively regulates their formation after initiation. Our data provide genetic evidence supporting the concept of early specification and progressive determination of anterior limb pattern., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.

Background: Studies have shown that the fracture plane of the classic metaphyseal lesion (CML) of infant abuse occurs in the region of the primary spongiosa, encompassing a radiodense fracture fragment customarily referred to as the "zone of provisional calcification" or ZPC. However, the zone of provisional calcification is defined differently in the pathology and the imaging literature, potentially impeding efforts to understand the fundamental morphological features of the classic metaphyseal lesion., Objective: We systematically correlated micro-CT data with histology in piglets to explore the differing definitions of the zone of provisional calcification and to elucidate the anatomical basis for divergent definitions., Materials and Methods: The distal tibias of five normal fetal piglets were studied postmortem. The specimens were resected and imaged with digital radiography (50 μm resolution) and micro-CT (45 μm(3) isotropic resolution). Image processing techniques were applied to the micro-CT data for visualization and data analysis. The resected tissue specimens were then processed routinely and the light microscopic features were correlated with the imaging findings., Results: The longitudinal dimension of the radiologic zone of provisional calcification is greater than the histological ZPC, and these dimensions are statistically distinct (P < 0.0002). The radiologic zone of provisional calcification consists of two adjoining mineralized discoid regions that span the chondro-osseous junction-a thick discoid region that encompasses the densest region of the primary spongiosa, and a thin discoid region (corresponding to the histological ZPC) that is situated in the base of the physis adjacent to the metaphysis., Conclusion: The correlation of the normal histology and micro-CT appearance of this dynamic and complex region provides an anatomical foundation upon which a deeper appreciation of the morphology of the classic metaphyseal lesion can be built.

The current study was undertaken to describe the main histological development stages of long bones (tibia and femur) from Japanese quail (Coturnix coturnix japonica) embryos. Whole Limbs or just tibia and femur of fifty Japanese quail embryos of different ages were fixed and embedded in paraffin or Spurr's resin. Paraffin and semi-thin, respectively, were undertaken and examined with light microscopy. Limb bud was established at day 5 of incubation. Mesenchymal cells differentiated into chondrocytes forming a cartilage template in the position of the future tibia and femur at day 6 of incubation. At day 7 of incubation, the cartilage template enlarged and had the shape and position of the future tibia and femur. At day 8, central chondrocytes underwent hypertrophy and were surrounded by a periosteal bone collar. Cellular and vascular invasion from the bone collar into the central zone of the cartilage template, cartilage resorption and formation of marrow tunnel and finally peripheral calcification was seen. Vascular cartilage canals penetrating the epiphysis were observed at day 9 and the canals gradually increased in thickness and number toward the hatching day. Articular epiphyseal growth cartilage with resting, proliferative and hypertrophic zones was clearly established by day 10 of incubation. After 17 days of incubation, the zonation of the articular epiphyseal cartilage were much clear, many cartilage canals were present within the epiphyses. In epiphyses of tibia but not femur, foci of chondrocytes hypertrophy were noticed close to the cartilage canals. The current study timed the main histological sequences of development of tibia and femur of embryonic quail.

There is compelling in vivo evidence from reports on human genetic mutations and transgenic mice that some microRNAs (miRNAs) play an important functional role in regulating skeletal development and growth. A number of published in vitro studies also point toward a role for miRNAs in controlling chondrocyte gene expression and differentiation. However, information on miRNAs that may regulate a specific phase of chondrocyte differentiation (i.e. production of progenitor, differentiated or hypertrophic chondrocytes) is lacking. To attempt to bridge this knowledge gap, we have investigated miRNA expression patterns in human embryonic cartilage tissue. Specifically, a developmental time point was selected, prior to endochondral ossification in the embryonic limb, to permit analysis of three distinct populations of chondrocytes. The location of chondroprogenitor cells, differentiated chondrocytes and hypertrophic chondrocytes in gestational day 54-56 human embryonic limb tissue sections was confirmed both histologically and by specific collagen expression patterns. Laser capture microdissection was utilized to separate the three chondrocyte populations and a miRNA profiling study was carried out using TaqMan® OpenArray® Human MicroRNA Panels (Applied Biosystems®). Here we report on abundantly expressed miRNAs in human embryonic cartilage tissue and, more importantly, we have identified miRNAs that are significantly differentially expressed between precursor, differentiated and hypertrophic chondrocytes by 2-fold or more. Some of the miRNAs identified in this study have been described in other aspects of cartilage or bone biology, while others have not yet been reported in chondrocytes. Finally, a bioinformatics approach was applied to begin to decipher developmental cellular pathways that may be regulated by groups of differentially expressed miRNAs during distinct stages of chondrogenesis. Data obtained from this work will serve as an important resource of information for the field of cartilage biology and will enhance our understanding of miRNA-driven mechanisms regulating cartilage and endochondral bone development, regeneration and repair.

Objective: Syndecan 4, a heparan sulfate proteoglycan, has been associated with osteoarthritis. The present study was undertaken to analyze the functional role of syndecan 4 in endochondral ossification of mouse embryos and in adult fracture repair, which, like osteoarthritis, involves an inflammatory component., Methods: Sdc4 promoter activity was analyzed in Sdc4(-/-) lacZ-knockin mice, using β-galactosidase staining. Endochondral ossification in embryos from embryonic day 16.5 was assessed by histologic and immunohistologic staining. Bone fracture repair was analyzed in femora of adult mice on days 7 and 14 postfracture. To evaluate Sdc2 and Sdc4 gene expression with and without tumor necrosis factor α (TNFα) and Wnt-3a stimulation, quantitative real-time polymerase chain reaction was performed., Results: In Sdc4(-/-) lacZ-knockin animals, syndecan 4 promoter activity was detectable at all stages of chondrocyte differentiation, and Sdc4 deficiency inhibited chondrocyte proliferation. Aggrecan turnover in the uncalcified cartilage of the epiphysis was decreased transiently in vivo, but this did not lead to a growth phenotype at birth. In contrast, among adult mice, fracture healing was markedly delayed in Sdc4(-/-) animals and was accompanied by increased callus formation. Blocking of inflammation via anti-TNFα treatment during fracture healing reduced these changes in Sdc4(-/-) mice to levels observed in wild-type controls. We analyzed the differences between the mild embryonic and the severe adult phenotype, and found a compensatory up-regulation of syndecan 2 in the developing cartilage of Sdc4(-/-) mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt-3a in vitro led to increased expression of syndecan 2, while stimulation with TNFα resulted in up-regulation of syndecan 4 but decreased expression of syndecan 2. TNFα stimulation reduced syndecan 2 expression and increased syndecan 4 expression even in the presence of Wnt-3a, suggesting that inflammation has a strong effect on the regulation of syndecan expression., Conclusion: Our results demonstrate that syndecan 4 is functionally involved in endochondral ossification and that its loss impairs fracture healing, due to inhibition of compensatory mechanisms under inflammatory conditions., (Copyright © 2013 by the American College of Rheumatology.)

Sources of Zn, Mn, and Cu (IZMC) as sulfates or as amino acid complexes (OZMC) were used to supplement Cobb 500 breeder hen diets. Experimental treatments consisted of diets supplemented with 1) 100, 100, and 10 mg/kg of Zn, Mn, and Cu, respectively, from IZMC (control); 2) 60, 60, and 3 mg/kg of Zn, Mn, and Cu, respectively, from IZMC plus 40, 40, and 7 mg/kg of Zn, Mn, and Cu, respectively, from OZMC (ISO); and 3) a diet with 100, 100, and 10 mg/kg of Zn, Mn, and Cu, respectively, from IZMC as in control plus 40, 40, and 7 mg/kg of supplemental Zn, Mn, and Cu from OZMC (on top). Ten replications of 20 females and 2 males were used per treatment. Eggs from breeders at 30, 40, 50 and 60 wk of age were incubated, and 5 embryos per replicate were collected at 10 (E10), 14 (E14), and 18 (E18) d of incubation. Midshaft width and calcification were measured for left tibia and femur stained with Alcian Blue and Alizarin Red S. At hatch, the left tibia of 5 chicks per replicate was sampled for histological evaluation of the diaphysis and distal epiphysis. Feeding the ISO treatment compared with the control diet increased the Zn (P < 0.05) but not Mn and Cu content of the yolk and albumen blend. At E14, the ISO and on-top treatments had a trend to increase tibia calcification at the rates of 1.6 and 1%, respectively (P < 0.1). The E18 ISO and on-top treatments had 2% thicker tibia compared with the control, regardless of hen age (P < 0.05). Also, at E18, calcification of tibia and femur was higher from hens fed the on-top treatment (P < 0.05). The chicks from the ISO and on-top groups had increased tibia moment of inertia (P < 0.01) at day of hatch. Broiler breeder hens consuming OZMC associated with IZMC produced embryos and hatching chicks with improvements in selected bone mineralization parameters.

Detrimental effects of aflatoxin B1 (AFB1) on the embryonic development of broiler tibia and its proximal growth plate were determined by means of histological, histometric and immunohistochemical methods. For this, 420 fertile eggs from parent stocks of Ross 308 broiler chickens were divided into five groups according to the proposed treatment: a control untreated group, a group injected with 30% ethanol and three further groups to be injected with 5, 15 or 40 ng AFB1. The eggs were injected into the air space prior to incubation. Five eggs from each group were opened at 9, 11, 13, 17, 19 and 21 days of incubation and tibial tissue samples were removed, dissected of muscle and connective tissues, and processed by means of routine histological techniques. The cell proliferation rate of the epiphyseal growth plate cells was determined by immunohistochemical assay of proliferating cell nuclear antigen (PCNA) expression. The results showed that both proliferative and hypertrophic zones narrowed significantly (P<0.05), when compared with the controls, in all of the AFB1-treated groups whereas the transitional zone thickened, especially in the group given 40 ng AFB1 group. The PCNA positivity indices of 15 and 40 ng AFB1-treated groups were significantly higher (P<0.05) on days 11, 13, 17, 19 and 21 of incubation. It was concluded that in ovo-administered AFB1 adversely affected embryonic development of the tibial growth plate, and that affected hatched broilers might also be more susceptible to skeletal disorders during growth.

The article presents the results of a comparative histological investigation of skeletal bones genesis in Japanese quail embryos developed in the spaceflight microgravity (space group) and laboratory (control group). Total preparations of 4-day-old embryos from both groups demonstrated clearly that the cartilaginous anlage of the femoral bone had central, dyaphisial, 2 epiphysial and 2 proliferation zones. By day 7 of embryogenesis, cartilaginous anlages had grown in size in both groups due to intensive chondrocytes multiplication and gain in the intercellular substance mass. Tibial cuff in space embryos measured half and femoral cuff was 2.3 times smaller in comparison with these parameters in the control group. In addition, intensity of chondrocyte multiplication was reduced Histological profiles of the femur and tibia in 10-day old embryos of the control pointed to enhancement of osteogenesis. The metaphysis zone contained distinct mitosis figures on different stages of division. Bone deposition could be seen below the peristoma. The osteogenesis cuff spread up to the femoral anlage metaphysis; cartilage was calcined. Space embryos display retard osteogenesis. There were ingrown blood vessels in the region of cartilage destruction; however, vessels grown in the periosteum were less in number as compared with the laboratory control. Also, the perichondral ossification layer was considerably thinner, whereas the osseous cuff was 1.3 and 1.45 times shorter in the femur and tibia, respectively. To sum up, the histological investigation of bones from 4-, 7- and 10- day old Japanese quail embryos demonstrated retardation of osteogenesis in the conditions of microgravity.

Cartilage oligomeric matrix protein (COMP), a secreted glycoprotein synthesized by chondrocytes, regulates proliferation and type II collagen assembly. Mutations in the COMP gene cause pseudoachondroplasia and multiple epiphyseal dysplasia. Previously, we have shown that expression of D469del-COMP in transgenic mice causes intracellular retention of D469del-COMP, thereby recapitulating pseudoachondroplasia chondrocyte pathology. This inducible transgenic D469del-COMP mouse is the only in vivo model to replicate the critical cellular and clinical features of pseudoachondroplasia. Here, we report developmental studies of D469del-COMP-induced chondrocyte pathology from the prenatal period to adolescence. D469del-COMP retention was limited prenatally and did not negatively affect the growth plate until 3 weeks after birth. Results of immunostaining, transcriptome analysis, and qRT-PCR suggest a molecular model in which D469del-COMP triggers apoptosis during the first postnatal week. By 3 weeks (when most chondrocytes are retaining D469del-COMP), inflammation, oxidative stress, and DNA damage contribute to chondrocyte cell death by necroptosis. Importantly, by crossing the D469del-COMP mouse onto a Chop null background (Ddit3 null), thereby eliminating Chop, the unfolded protein response was disrupted, thus alleviating both D469del-COMP intracellular retention and premature chondrocyte cell death. Chop therefore plays a significant role in processes that mediate D469del-COMP retention. Taken together, these results suggest that there may be an optimal window before the induction of significant D469del-COMP retention during which endoplasmic reticulum stress could be targeted., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF) plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/-) mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/-) mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/-) mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/-) mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/-) mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/-) mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/-) mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/-) mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.

Tibia and femur osteogenesis was studied in embryonic lower limbs developed in microgravity. The maximal difference in ash content of shell in the flight and control groups was registered on days 4, 10 and 14 amounting to 10.27; 9.56 and 12.95% respectively. Shell analysis for calcium showed the largest difference between the groups on day 4 (8.94 mg). However, this difference was not seen already on days 14 and 16. Hence, according to the results of the investigation, although shell calcium utilization and osteogenesis in flight embryos of the Japanese quail were retarded, by the time of hatching the morphological and chemical parameters of the lower limb bones were essentially same as of the ground controls.

Objective: Skeletal abnormalities are one of the hallmarks of growth delay during gestation. The aim of this study was to determine changes induced by leptin in skeletal growth and development in a rat model of intrauterine growth retardation (IUGR) and to elucidate the possible underlying mechanisms., Methods: Intrauterine growth retardation was induced prepartum and the effects of leptin to mothers prenatally or to offspring postnatally were studied. Radii were harvested and tested mechanically and structurally. Tibias were evaluated for growth-plate morphometry., Results: On day 40 postpartum, total bone length and mineral density and tibial growth-plate width and numbers of cells within its zones of offspring treated with leptin were significantly greater than in the control group., Conclusion: Postnatal leptin administration in an IUGR model improves the structural properties and elongation rate of bone. These findings could pave the way to preventing some phenotypic presentations of IUGR., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Summary: This study compared bone status between Chinese and Caucasian infants at birth, showing that Chinese neonates have lower tibial speed of sound, which is influenced by gender, gestational age, season of birth, and maternal vitamin D status. The effects of these factors on fetal bone development were discussed., Introduction: We compared the differences of speed of sound (SOS) accessed by quantitative ultrasound between Chinese and Caucasian infants at birth and explored the relationship between the concentrations of serum 25-hydroxyvitamin D [25(OH)D] and bone SOS in maternal-infant pairs., Methods: SOS for the tibial bone was measured at birth in 267 Chinese infants. We used the Z-scores for the direct comparisons which were available from the instrument based data of gender and age-matched Caucasian peers. The concentrations of serum 25(OH)D and bone SOS in 32 maternal-infant pairs were measured at birth in winters., Results: the Chinese infants had lower SOS demonstrated by the Z-scores. Significant differences of SOS and Z-scores were found between genders, gestational ages, birth weight, and seasons of birth. The differences of Z-scores negatively decreased with gestational age, suggesting that the bone status of Chinese infants lags behind that of the Caucasian infants during the last trimester of pregnancy in utero. The tibial SOS of infants born in winters was 2.0% higher than those born in summers after adjustment. The infant SOS correlated with maternal serum 25(OH)D (r = 0.399, P = 0.024) and infant serum 25(OH)D (r = 0.394, P = 0.026)., Conclusions: Chinese neonates have lower SOS which is influenced by gender, gestational age, season of birth, and maternal vitamin D status. It is inferred that, in pace with gestational age, race and gender effects on fetal bone development are modified by materno-fetal vitamin D status.

Galanin exerts tonic inhibition of nociceptive input to the central nervous system. Recently, this peptide was demonstrated in several neuronal and non-neuronal structures in bones and joints. In this study, the time of appearance and topographic localization of galanin-containing nerve fibres in bone were studied in rats from gestational day 16 (GD16) to postnatal day 21 (PD21). The tibia was chosen as a model of developing long bone and indirect immunofluorescence combined with confocal laser scanning microscopy was used to identify galanin-immunoreactive (GAL-IR) nerve fibres. The earliest, sparse GAL-IR fibres were observed on GD21 in the perichondrium of both epiphyses and in the periosteum of the diaphysis. From PD1 onwards, GAL-IR fibres were also seen in the bone marrow cavity and in the region of the inter-condylar eminence of the knee joint. Intramedullary GAL-IR fibres in proximal and distal metaphyses appeared around PD1. Some of them accompanied blood vessels, although free fibres were also seen. GAL-IR fibres located in the cartilage canals of both epiphyses were observed from PD7, in the secondary ossification centres from PD10 and in the bone marrow of both epiphyses from PD14. The time course and localization of galanin-containing nerve fibres resemble the development of substance P- and CGRP-expressing nerve fibres, thus suggesting their sensory origin.

This study evaluated the effects of zinc on skeletal development during fetal development in pregnant ICR mice fed a zinc-deficient (3 mg/kg) or zinc-adequate (30 mg/kg) diet. We also included a group pair-fed with the zinc-deficient group to control for decreased appetite due to zinc deficiency. Developing fetuses at embryonic day 18.5 were removed by cesarean section, and the skeletal development was evaluated by histological analysis as well as by body weight and longitudinal growth measurement. Reduced maternal food intake in the zinc-deficient and pair-fed groups resulted in a marked and significant (P < .05) decrease in fetal weight compared to that of the zinc-adequate group. However, fetal length retardation in the pair-fed group was less marked than in the zinc-deficient group, suggesting that reduced supply of zinc from maternal circulation may play a role in longitudinal growth through skeletal development. The fetal developing tibia of the zinc-deficient group showed marked shortening of diaphysis and a mild narrowing of the hypertrophic chondrocyte zone width with increased osteoclast number, but there was no influence on the mineralization of bone matrix. This may be the result of reduced activation of osteoblasts and maturation of chondrocytes with increased osteoclastic activity, suggesting that zinc deficiency during the fetal development has a greater impact on the matrix formation of bone than the mineralization of bone matrix.

Previous work has reported the results of a multidisciplinary effort producing a proof-of-concept on the use of pectic polysaccharides in the surface modification of medical devices. This study was designed to learn more about the capability of engineered rhamnogalacturonan-I (RG-I) fractions of apple pectin to control bone cell and macrophage behavior. Thermanox or polystyrene Petri dishes were surface modified with two different modified hairy regions (MHRs) obtained by different enzymatic liquefaction processes of apples differing in relative amounts and lengths of their neutral side chains: (long-haired) MHR-alpha and (short-haired) MHR-B. Bone explants from 14-day-old chick embryos were cultured for 14 days on both pectic substrata. MHR-B promoted cell migration and differentiation, MHR-alpha did not. On MHR-alpha, J774.2 macrophages grew well, their percentage in G1 phase was decreased and in S phase increased, and they did not secrete either proinflammatory-cytokines or nitrites. Contrasting results were gained from macrophages on MHR-B, except for nitrite secretion. Thus, we conclude that coatings from tailored pectins show different biological activities in vitro and are potential innovative candidates for improving the biocompatibility of medical devices in various applications.

Temperature (TEM) and O(2) concentrations during the plateau stage of oxygen consumption are known to affect yolk utilization, tissue development, and thyroid metabolism in turkey embryos. Three experiments were conducted to evaluate these incubation effects on long bone development. Fertile eggs of Nicholas turkeys were used. In each trial, standard incubation conditions were used to 24 d, when the eggs containing viable embryos were randomly divided into 4 groups. Four experimental cabinets provided 4 TEM (36, 37, 38, or 39 degrees C) or 4 O(2) concentrations (17, 19, 21, or 23% O(2)). In the third experiment, 2 temperatures (36 and 39 degrees C) and 2 O(2) concentrations (17 and 23%) were evaluated in a 2 x 2 factorial design. Body and residual yolk weights were obtained. Both legs were dissected, and shanks, femur, and tibia weights, length, and thickness were recorded. Relative asymmetry of each leg section was calculated. Chondrocyte density was evaluated in slides stained with hematoxylin and eosin. Immunofluorescence was used to evaluate the presence of collagen type X and transforming growth factor beta. Hot TEM caused reduction of tibia weights and increase of shank weight when compared with cool TEM. The lengths of femur, tibia, and shanks were reduced by 39 degrees C. The relative asymmetry of leg weights were increased at 38 and 39 degrees C. Poult body and part weights were not affected by O(2) concentrations, but poults on 23% O(2) had bigger shanks and heavier tibias than the ones on 17% O(2). High TEM depressed the fluorescence of collagen type X and transforming growth factor beta. The O(2) concentrations did not consistently affect the immunofluorescence of these proteins. The chondrocyte density was affected by TEM and O(2) in resting and hypertrophic zones. In the third experiment, high TEM depressed BW, leg muscle weights, and shank length. Low O(2) reduced tibia and shanks as a proportion of the whole body. We concluded that incubation conditions affect long bone development in turkeys.

Reasons for Performing Study: The earliest osteochondrosis (OC) microscopic lesion reported in the literature was present in the femorotibial joint of a 2-day-old foal suggesting that OC lesions and factors initiating them may arise prior to birth., Objective: To examine the developing equine epiphysis to detect histological changes that could be precursors to OC lesions., Methods: Osteochondral samples from 21 equine fetuses and 13 foals were harvested from selected sites in the scapulohumeral, humeroradial, metacarpophalangeal, femoropatellar, femorotibial, tarsocrural and metatarsophalangeal joints. Sections were stained with safranin O and picrosiruis red to assess cartilage changes and structural arrangement of the collagen matrix., Results: Extracellular matrix changes observed included perivascular areas of paleness of the proteoglycan matrix associated with hypocellularity and, sometimes, necrotic chondrocytes. These changes were most abundant in the youngest fetuses and in the femoropatellar/femorotibial (FP/FT) joints. Indentations of the ossification front were also observed in most specimens, but, most frequently, in scapulohumeral and FP/FT joints. A cartilage canal was almost always present in these indentations. The vascular density of the cartilage was higher in the youngest fetuses. In these fetuses, the most vascularised joints were the metacarpo- and metatarsophalangeal joints but their cartilage canals regressed quickly. After birth, the most vascularised cartilage was present in the FP/FT joint. Articular cartilage differentiated into 4 zones early in fetal life and the epiphyseal cartilage also had a distinct zonal cartilage structure. A striking difference was observed in the collagen structure at the junction of the proliferative and hypertrophic zones where OCD lesions occur., Conclusion: Matrix and ossification front changes were frequently observed and significantly associated with cartilage canals suggesting that they may be physiological changes associated with matrix remodelling and development. The collagen structure was variable through the growing epiphysis and a differential in biomechanical properties at focal sites may predispose them to injury.

Objective: The purpose of this study was to establish a new reference growth chart of calf muscle biometric measurements throughout gestation in normal singleton pregnancies., Methods: A prospective cohort study was designed. One hundred pregnant women were included in the study and assessed by 3-dimensional sonography. Excluded were those with multiple pregnancies, congenital anomalies, abnormal karyotypes, and polyhydramnios or oligohydramnios. Three-dimensional multiplanar sonographic images were used to measure the calf muscles: soleus, gastrocnemius, popliteal, peroneus longus, and tibialis posterior., Results: Calf muscle widths increased with increasing gestational age (R(2) = 0.857; P < .0001), and the ratio between calf muscles and the tibia increased as well (R(2) = 0.356; P = .001)., Conclusions: The data provide a nomogram of calf width with a direct correlation between muscle growth and gestational age. This nomogram can offer a basis for normal calf development and may assist in distinguishing between different etiologies leading to clubfoot and other joint contractures associated with calf atrophy.

Carbonic anhydrase II (CAII)-deficient mice were created to study the syndrome of CAII deficiency in humans including osteopetrosis, renal tubular acidosis, and cerebral calcification. Although CAII mice have renal tubular acidosis, studies that analyzed only cortical bones found no changes characteristic of osteopetrosis. Consistent with previous studies, the tibiae of CAII-deficient mice were significantly smaller than those of wild-type (WT) mice (28.7 +/- 0.9 vs. 43.6 +/- 3.7 mg; p < 0.005), and the normalized cortical bone volume of CAII-deficient mice (79.3 +/- 2.2%) was within 5% of that of WT mice (82.7 +/- 2.3%; p < 0.05), however, metaphyseal widening of the tibial plateau was noted in CAII-deficient mice, consistent with osteopetrosis. In contrast to cortical bone, trabecular bone volume demonstrated a nearly 50% increase in CAII-deficient mice (22.9 +/- 3.5% in CAII, compared to 15.3 +/- 1.6% in WT; p < 0.001). In addition, histomorphometry demonstrated that bone formation rate was decreased by 68% in cortical bone (4.77 +/- 1.65 microm3/microm2/day in WT vs. 2.07 +/- 1.71 microm3/microm2/day in CAII mice; p < 0.05) and 55% in trabecular bone (0.617 +/- 0.230 microm3/microm2/day in WT vs. 0.272 +/- 0.114 microm3/microm2/day in CAII mice; p < 0.05) in CAII-deficient mice. The number of osteoclasts was significantly increased (67%) in CAII-deficient mice, while osteoblast number was not different from that in WT mice. The metaphyseal widening and changes in the trabecular bone are consistent with osteopetrosis, making the CAII-deficient mouse a valuable model of human disease.

We previously observed using cultured tibiotarsal long-bone rudiments from which the perichondrium (PC) and periosteum (PO) was removed that the PC regulates cartilage growth by the secretion of soluble negative regulatory factors. This regulation is "precise" in that it compensates exactly for removal of the endogenous PC and is mediated through at least three independent mechanisms, one of which involves a response to TGF-beta. PC cell cultures treated with 2 ng/ml TGF-beta1 produced a conditioned medium which when added to PC/PO-free organ cultures effected precise regulation of cartilage growth. In the present study, we have investigated the possibility that TGF-beta itself might be the negative regulator which is produced by the PC cells in response to their treatment with TGF-beta1. Using a TGF-beta responsive reporter assay, we determined that PC cell cultures, when treated with 2 ng/ml or greater exogenous TGF-beta1, produce 300 pg/ml of active TGF-beta. Then we observed that this concentration (300 pg/ml) of active TGF-beta1, when added to PC/PO-free tibiotarsal organ cultures, effected precise regulation of cartilage growth, whereas concentrations of TGF-beta1 either greater or less than 300 pg/ml produced abnormally small cartilages. These results suggest that one mechanism by which the PC effects normal cartilage growth is through the production of a precisely regulated amount of TGF-beta which the PC produces in response to treatment with exogenous TGF-beta itself.

Unlabelled: Mandibular and systemic bone loss are poorly associated. We compared the effect of isocaloric protein undernutrition and/or ovariectomy on BMD and microstructure of mandibular alveolar and proximal tibia sites in adult rats. Mandibular bone was significantly less affected., Introduction: Whether mandibular bone and axial or peripheral skeleton respond similarly to systemic bone loss remains a subject of controversy. We have previously shown that mechanical loading during mastication influences bone mass and architecture of the mandibular alveolar bone. Isocaloric protein undernutrition and ovariectomy are known to cause bone loss and deterioration of bone microarchitecture at various axial and peripheral skeletal sites. We studied how the mandible, which is subjected to heavy, abrupt, and intermittent forces during mastication, responds to low-protein intake and/or ovariectomy and compared this response to that of the proximal tibia in adult rats., Materials and Methods: Forty-four 6-month-old female Sprague-Dawley rats underwent transabdominal ovariectomy (OVX; n=22) or sham operation (n=22) and were pair-fed isocaloric diets containing either 15% or 2.5% casein (sham 15%, n=11; sham 2.5%, n=11; OVX 15%, n=11; and OVX 2.5%, n=11) for 16 weeks. BMD and bone microarchitecture parameters (e.g., bone volume fraction [BV/TV] and trabecular thickness and number) of the mandible and the proximal tibia were measured at the end of the experiment using DXA and microCT., Results: Mandibular alveolar bone was negatively influenced by both protein undernutrition and OVX, but to a significantly lesser extent than the proximal tibia. In sham-operated animals, low-protein intake led to a 17.3% reduction of BV/TV in the mandible and 84.6% in the tibia (p<0.001). In normal protein diet-fed animals, OVX led to a reduction of BV/TV of 4.9% in the mandible but 82% in the tibia (p<0.001). In the mandible, protein undernutrition resulted in thinner trabeculae (p<0.05), whereas OVX led to a reduction of trabecular number (p<0.05)., Conclusions: Mandibular alveolar bone was found to be less sensitive to either protein undernutrition or OVX than the proximal tibia spongiosa. We hypothesize that the mechanical loading of the alveolar process during mastication may protect the alveolar bone from the detrimental effects observed in other skeletal sites, such as the proximal tibia. Morphological and embryological differences between the two skeletal sites might also play a role.

The transcription factor nuclear factor E2 p45-related factor 2 (Nrf2) forms heterodimers with small musculoaponeurotic fibrosarcoma (Maf) proteins for the selective recognition of the antioxidant responsive element on target genes, followed by the regulation of gene expression of phase II detoxifying enzymes as well as oxidative-stress-inducible proteins in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of chondrocyte differentiation as well as the expression pattern of Nrf2 in cartilage. In tibia from embryonic mice at E15.5, Nrf2 mRNA expression was restricted to both proliferating and pre-hypertrophic chondrocytes, with few signals in early and late hypertrophic chondrocytes expressing both type X collagen and osteopontin. On in situ hybridization analysis of tibia from neonatal mice at 1 day after birth, by contrast, Nrf2 was expressed in all chondrocytic layers in addition to osteoblasts attached to cancellous bone. In pre-chondrogenic cell line ATDC5 cells, furthermore, expression of Nrf2 mRNA was also confirmed together with mRNA expression of the Kelch-like ECH associating protein 1 and small Maf proteins. In ATDC5 cells stably transfected with Nrf2, significant inhibition was seen in the differentiation-dependent induction of alkaline phosphatase and increase in the Alcian blue staining intensity. Furthermore, stable overexpression of Nrf2 significantly decreased mRNA expression of several chondrocyte differentiation markers such as type II collagen, type X collagen and osteopontin. These data suggest that Nrf2 may be a negative regulator of the cellular differentiation toward maturation in chondrocytes.

Background: Pregnancy-associated plasma protein-A (PAPP-A) is produced by the embryo and placenta during pregnancy, and its maternal serum concentrations are related to subsequent fetal growth. Evidence from animal models and in vitro experiments suggests that PAPP-A is particularly involved in the regulation of bone development. The aim of this study was to assess the correlation between late first trimester fetal bone length and maternal serum levels of PAPP-A., Methods: In a cross-sectional observational study, ultrasound measurements of fetal long bones and fluorimetric immunoassays for maternal serum PAPP-A were performed in 514 singleton pregnancies at 10-14 weeks of gestation., Results: There were 501 uncomplicated pregnancies. There were significant correlations between PAPP-A values and length of humerus, femur and tibia [r values 0.12 (P = 0.01), 0.11 (P = 0.01) and 0.10 (P = 0.03), respectively]. The association with the length of ulna and foot did not reach statistical significance (r values 0.08 and -0.03, respectively)., Conclusions: Maternal serum PAPP-A levels at 10-14 weeks of gestation are significantly associated with the length of fetal long bones such as humerus, femur and tibia. This provides further evidence that PAPP-A may be involved in the regulation of bone development.

Cartilage canals have been extensively investigated, in particular as to their mode of formation, morphologic distribution, function and fate. We studied the morphological pattern of the cartilage canals of the right upper chondroepiphysis of the tibia of chick embryos, Hamburger-Hamilton stages 35-42, in serial sagittal wax sections and in reconstructions made with AutoCAD software. The spatial arrangement of the canals is presented in a series of drawings made according to computerized images. The canals were penetrated from three distinct surfaces: the anterior and superior surfaces of the tubercle and the posterior surfaces of the medial and lateral condyles. Immediately after entering, nearly all the canals were extended toward the medial or lateral aspects of the chondroepiphysis, from which no canals took their origin. All of the cartilage canals connected with the perichondrium, and their branching did not follow a specific pattern. The condylar canals did not unite with the tubercular ones.

Purpose: This study was conducted to verify the predictive value of epiphyseal ossification center measurements in estimating gestational age., Methods: Women with singleton pregnancies of 30-40 weeks gestation (n = 377) were enrolled in this prospective study. The distal femoral, proximal tibial, and proximal humeral ossification centers were identified and measured. A nomogram of fetal bone development was created using the sum of the three diameters., Results: Gestational age correlated well with the diameters of the distal femoral and the proximal tibial epiphyseal ossification centers but even better with the sum of the three ossification centers. Positive predictive values of the fetus having gestational age of at least 37 weeks when the sum of the three centers was 7, 11, and 13 mm were 82%, 94%, and 100%, respectively. A nomogram was created using the sum of the ossification centers for 30-40 weeks' gestational age., Conclusions: Ultrasonographic visualization of the epiphyses ossification centers may be a useful marker of fetal gestational age., (Copyright 2005 Wiley Periodicals, Inc.)

The development of sensory innervation in long bones was investigated in rat tibia in fetuses on gestational days (GD) 16-21 and in neonates and juvenile individuals on postnatal days (PD) 1-28. A double immunostaining method was applied to study the co-localization of the neuronal growth marker growth-associated protein 43 (GAP-43) and the pan-neuronal marker protein gene product 9.5 (PGP 9.5) as well as that of two sensory fibre-associated neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP). The earliest, not yet chemically coded, nerve fibres were observed on GD17 in the perichondrium of the proximal epiphysis. Further development of the innervation was characterized by the successive appearance of nerve fibres in the perichondrium/periosteum of the shaft (GD19), the bone marrow cavity and intercondylar eminence (GD21), the metaphyses (PD1), the cartilage canals penetrating into the epiphyses (PD7), and finally in the secondary ossification centres (PD10) and epiphyseal bone marrow (PD14). Maturation of the fibres, manifested by their immunoreactivity for CGRP and SP, was visible on GD21 in the epiphyseal perichondrium, the periosteum of the shaft and the bone marrow, on PD1 in the intercondylar eminence and the metaphyses, on PD7 in the cartilage canals, on PD10 in the secondary ossification centres and on PD14 in the epiphyseal bone marrow. The temporal and topographic pattern of nerve fibre appearance corresponds with the development of regions characterized by active mineralization and bone remodelling, suggesting a possible involvement of the sensory innervation in these processes.

Background: Little is known about the developmental changes associated with tibial ray deficiencies. The aim of this study was to detect cell death, proliferation, and gene expression that result in tibial ray deficiencies., Methods: We induced tibial ray deficiencies in rat embryos using a teratogenic agent (busulfan) and observed the developmental changes in 1126 hindlimbs. We performed Nile blue staining, whole mount in situ hybridization for fibroblast growth factor 8 (Fgf8), bone morphogenetic protein 4 (Bmp4) and Sonic hedgehog (Shh), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and assessment of cell proliferation by 5-bromo-2'-deoxy-uridine (BrdU)/anti-BrdU immunohistochemistry., Results: In situ hybridization showed reductions in Fgf8 and Bmp4 expression. Histological examination showed a delay of mesenchymal condensation, increased mesenchymal cell death, decreased mesenchymal cell proliferation, and a reduction in the number of mesenchymal cells. These abnormalities may cause hypoplasia of the limb. Bmp4 expression was markedly reduced in the anterior mesenchyme. Shh was expressed in the posterior mesenchyme. We suggest that the posterior skeletal elements may be fully formed owing to Shh expression, but the anterior skeletal elements may be underdeveloped owing to an intense reduction of Bmp4 expression in the anterior mesenchyme, causing hypoplasia of the tibial ray., Conclusions: The combined effects of increased cell death, decreased cell proliferation, reduction of Fgf8 expression, and intense reduction of Bmp4 expression in the anterior mesenchyme may play an important role in the development of tibial ray deficiency induced by busulfan., ((c) 2005 Wiley-Liss, Inc.)

We previously reported that C-type natriuretic peptide (CNP) stimulates endochondral ossification and corrects the reduction in body length of achondroplasia model mouse with constitutive active fibroblast growth factor receptor 3 (FGFR-3). In order to examine the interaction between CNP and FGFR-3, we studied intracellular signaling by using ATDC5 cells, a mouse chondrogenic cell line, and found that FGF2 and FGF18 markedly reduced CNP-dependent intracellular cGMP production, and that these effects were attenuated by MAPK inhibitors. Western blot analysis demonstrated that the level of GC-B, a particulate guanylyl cyclase specific for CNP, was not changed by treatment with FGFs. Conversely, CNP and 8-bromo-cGMP strongly and dose-dependently inhibited the induction of ERK phosphorylation by FGF2 and FGF18 without changing the level of FGFR-3, although they did not affect the phosphorylation of STAT-1. In the organ-cultured fetal mouse tibias, CNP and FGF18 counteracted on the longitudinal bone growth, and both the size and number of hypertrophic chondrocytes. The FGF/FGFR-3 pathway is known as the negative regulator of endochondral ossification. We found that FGFs inhibited CNP-stimulated cGMP production by disrupting the signaling pathway through GC-B while CNP antagonized the activation of the MAPK cascade by FGFs. These results suggest that the CNP/GC-B pathway plays an important role in growth plate chondrocytes and constitutes the negative cross talk between FGFs and the activity of MAPK. Our results may explain one of the molecular mechanisms of the growth stimulating action of CNP and suggest that activation of the CNP/GC-B pathway may be effective as a novel therapeutic strategy for achondroplasia.

Objective: The purpose of this study was to produce a method of predicting limb length discrepancy in utero., Methods: Using available databases, we divided the femoral and tibial lengths at term by the femoral and tibial lengths at each week of gestation for each percentile. The quotients represent coefficients (multipliers) of limb segment growth at each prenatal age., Results: We found the prenatal multipliers to be independent of race, percentile, and gender from as early as 12 weeks' gestation. The prenatal multipliers are alike for femur and tibia., Conclusions: The prenatal multiplier method allows for quick prediction of limb length discrepancy at term and at skeletal maturity from as early as 12 weeks' gestation. Future study is needed to validate this method clinically.