Discoid meniscus in human fetuses: A systematic review.

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.

Ultrasonographic assessment of fetal thyroid in Japan: thyroid circumference and distal femoral and proximal tibial ossification.

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.

Development of the Proximal-Anterior Skeletal Elements in the Mouse Hindlimb Is Regulated by a Transcriptional and Signaling Network Controlled by Sall4.

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.

Culturing and Measuring Fetal and Newborn Murine Long Bones.

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.

Prenatal growth map of the mouse knee joint by means of deformable registration technique.

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.

Three-dimensional growth of tibial shaft ossification in the human fetus: a digital-image and statistical analysis.

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 (R2 = 0.89) for its length, y = - 2.855 + 0.307 × age ± 0.009 (R2 = 0.96) for its proximal transverse diameter, y = - 0.758 + 0.153 × age ± 0.005 (R2 = 0.88) for its middle transverse diameter, y = - 1.844 + 0.272 × age ± 0.09 (R2 = 0.90) for its distal transverse diameter, y = - 40.263 + 0.258 × (age)2 ± 0.007 (R2 = 0.94) for its projection surface area, and y = - 287.996 + 1.186 × (age)2 ± 0.037 (R2 = 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.

Expression of Signaling Molecules Involved in Embryonic Development of the Insertion Site Is Inadequate for Reformation of the Native Enthesis: Evaluation in a Novel Murine ACL Reconstruction Model.

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.

Prenatal nicotine exposure retards osteoclastogenesis and endochondral ossification in fetal long bones in rats.

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 α4ß2-nicotinic acetylcholine receptor (α4ß2-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.

Glypican-6 promotes the growth of developing long bones by stimulating Hedgehog signaling.

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.

Cartilage canals in the distal intermediate ridge of the tibia of fetuses and foals are surrounded by different types of collagen.

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.

Statins do not inhibit the FGFR signaling in chondrocytes.

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.

Expression of CGRP, vasculogenesis and osteogenesis associated mRNAs in the developing mouse mandible and tibia.

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.

Systematic radiographic evaluation of tibial hemimelia with orthopedic implications.

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.

Maintenance of Bone Homeostasis by DLL1-Mediated Notch Signaling.

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.

Histological and histochemical evaluations on the effects of high incubation temperature on the embryonic development of tibial growth plate in broiler chickens.

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.

Spatial Change of Cruciate Ligaments in Rat Embryo Knee Joint by Three-Dimensional Reconstruction.

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.

Porous deproteinized bovine bone scaffold with three-dimensional localized drug delivery system using chitosan microspheres.

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.

Three-dimensional reconstruction of rat knee joint using episcopic fluorescence image capture.

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.

Effect of eggshell temperature throughout incubation on broiler hatchling leg bone development.

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.

Formation of proximal and anterior limb skeleton requires early function of Irx3 and Irx5 and is negatively regulated by Shh signaling.

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.