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Vasculogenesis (de novo formation of vessels) induced by endothelial progenitor cells (EPCs) is requisite for vascularized bone regeneration. However, there exist few available options for promoting vasculogenesis within artificial bone grafts except for exogenous EPC transplantation, which suffers from the source of EPC, safety, cost, and time concerns in clinical applications. This study aimed at endogenous EPC recruitment for vascularized bone regeneration by using a bioinspired EPC-induced graft. The EPC-induced graft was created by immobilizing two bioactive peptides, WKYMVm and YIGSR, on the surface of poly(ε-caprolactone) (PCL)/poliglecaprone (PGC) nanofibrous scaffolds via a polyglycolic acid (PGA)-binding peptide sequence. Remarkable immobilization efficacy of WKYMVm and YIGSR peptides and their sustained release (over 14 days) from scaffolds were observed. In vivo and in vitro studies showed robust recruitment of EPCs, which subsequently contributed to early vasculogenesis and ultimate bone regeneration. The dual-peptide-functionalized nanofibrous scaffolds proposed in this study provide a promising therapeutic strategy for vasculogenesis in bone defect repair.

Studies have demonstrated that scaffolds, a component of bone tissue engineering, play an indispensable role in bone repair. However, these scaffolds involving ex-vivo cultivated cells seeded have disadvantages in clinical practice, such as limited autologous cells, time-consuming cell expansion procedures, low survival rate and immune-rejection issues. To overcome these disadvantages, recent focus has been placed on the design of functionalized cell-free scaffolds, instead of cell-seeded scaffolds, that can reduplicate the natural self-healing events of bone fractures, such as inflammation, cell recruitment, vascularization, and osteogenic differentiation. New approaches and applications in tissue engineering and regenerative medicine continue to drive the development of functionalized cell-free scaffolds for bone repair. In this review, the self-healing processes were highlighted, and approaches for the functionalization were summarized. Also, ongoing efforts and breakthroughs in the field of functionalization for bone defect repair were discussed. Finally, a brief summery and new perspectives for functionalization strategies were presented to provide guidelines for further efforts in the design of bioinspired cell-free scaffolds.

The purpose of this study was to detect the correlation between axial scan orientation discrepancy and the knee anatomical parameters associated with patellofemoral instability during computed tomography (CT) scanning. CT scan data of 16 knees from 16 healthy subjects were retrieved. The reslicing processes were performed to simulate different axial scan orientations generated due to leg adduction/abduction during CT scanning. Eight parameters (tibial tubercle to trochlear groove [TT-TG], tibial tubercle to posterior cruciate ligament [TT-PCL], patellar tilt, lateral trochlear inclination, sulcus angle, trochlear depth, trochlear facet asymmetry, and trochlear condyle asymmetry) of five angles (neutral zero, 4/8 degrees of adduction and 4/8 degrees abduction) were quantified. All eight parameters showed varying degrees of deviations to the different axial scan orientations. TT-TG, patellar tilt, lateral trochlear inclination, and trochlear condyle asymmetry were relatively sensitive to axial scan orientation changes, whereas TT-PCL, sulcus angle, trochlear depth, and trochlear facet asymmetry were relatively insensitive. Although the sensitivities are various, surgeons should be aware of this situation and make necessary corrections.

Swallow or deglutition syncope is a relatively rare syndrome. It is a vagally mediated syncope induced by swallowing. Swallow syncope may occur in all age groups and, when diagnosed, is treatable. A woman, aged 60 years, presented with an episode of a syncopal attack associated with swallowing a sandwich. She had a 6-month history of recurrent episodes of lightheadedness while eating solid foods. Telemetry monitoring demonstrated several episodes of severe bradycardia and complete atrioventricular block with up to a 7.0 second pause associated with meals. Computed tomography of the head and neck revealed no significant findings, and barium esophagram was normal. Echocardiogram was within normal limits. Her symptoms resolved after permanent pacemaker placement. Herein, we review the diagnosis, mechanism, and management of swallow syncope.

The estrogen-related receptor alpha (ERRalpha) is an orphan member of the superfamily of nuclear hormone receptors expressed in tissues that preferentially metabolize fatty acids. Despite the molecular characterization of ERRalpha and identification of target genes, determination of its physiological function has been hampered by the lack of a natural ligand. To further understand the in vivo function of ERRalpha, we generated and analyzed Estrra-null (ERRalpha-/-) mutant mice. Here we show that ERRalpha-/- mice are viable, fertile and display no gross anatomical alterations, with the exception of reduced body weight and peripheral fat deposits. No significant changes in food consumption and energy expenditure or serum biochemistry parameters were observed in the mutant animals. However, the mutant animals are resistant to a high-fat diet-induced obesity. Importantly, DNA microarray analysis of gene expression in adipose tissue demonstrates altered regulation of several enzymes involved in lipid, eicosanoid, and steroid synthesis, suggesting that the loss of ERRalpha might interfere with other nuclear receptor signaling pathways. In addition, the microarray study shows alteration in the expression of genes regulating adipogenesis as well as energy metabolism. In agreement with these findings, metabolic studies showed reduced lipogenesis in adipose tissues. This study suggests that ERRalpha functions as a metabolic regulator and that the ERRalpha-/- mice provide a novel model for the investigation of metabolic regulation by nuclear receptors.

One action of retinoids in devel- opment is the regulation of Hox gene expression. Hoxd4 and Hoxb4 expression in the embryonic hindbrain is anteriorized by retinoic acid (RA) treatment of mid-gestation mouse embryos. Here we demonstrate that retinoic acid receptor beta (Rarb) deficient mice present only a partial ante- riorization of either Hoxd4 or Hoxb4 in response to RA treatment. Our results strongly suggest that RARb is a mediator of their RA-response, and reveal anteroposterior polarity within a single rhombomere (r). Additionally, we gener- ated mice doubly mutated for Hoxd4 and Rarb in an attempt to identify common morphogenetic pathways between these two genes. We conclude that there are no synergistic interactions be- tween Hoxd4 and Rarb in the specification of the cervical vertebrae. Dev Dyn 1999;215:96-107.

Classical endocrine studies have shown that steroid hormones are required for the maintenance of pregnancy and placental viability. The oestrogen-receptor-related receptor beta (ERR-beta) is an orphan member of the superfamily of nuclear hormone receptors. Although ERR-beta is homologous to the oestrogen receptor and binds the oestrogen response element, it is not activated by oestrogens. Expression of ERR-beta during embryogenesis defines a subset of extra-embryonic ectoderm that subsequently forms the dome of the chorion, suggesting that ERR-beta may be involved in early placental development. Homozygous mutant embryos generated by targeted disruption of the Estrrb gene have severely impaired placental formation, and die at 10.5 days post-coitum. The mutants display abnormal chorion development associated with an overabundance of trophoblast giant cells and a severe deficiency of diploid trophoblast. The phenotype can be rescued by aggregation of Estrrb mutant embryos with tetraploid wild-type cells, which contribute exclusively to extra-embryonic tissues. Our results indicate that ERR-beta has an important role in early placentation, and suggest that an inductive signal originating from or modified by the chorion is required for normal trophoblast proliferation and differentiation.

Abstract Several aspects of normal cardiovascular development require signaling by the vitamin A metabolite retinoic acid. We have previously established germ-line mutations in mice in the genes that encode the RARα1, RARβ, and RXRα retinoic acid receptors as a means of studying the function of these receptors in vivo. Although mutation of RXRα results in fetal ventricular defects, the RARα1 and RARβ mutations are apparently nonphenotypic in the heart and elsewhere. In this study, we have established and analyzed combinations of these receptor gene mutations. Malformations of the ventricular chamber (chamber hypoplasia and muscular ventricular septal defects), conotruncus (double-outlet right ventricle, transposition, and membranous ventricular septal defects), aortic sac (persistent truncus arteriosus and aorticopulmonary window), and aortic arch–derived arteries were recovered in various combinations of the RARα1, RARβ, and RXRα gene mutations. Depending on the combination of receptor mutations, selective defects were obtained in specific cardiovascular compartments, suggestive of differential expression or function of each receptor within domains of the developing heart.

Mice with targeted disruptions in retinoic acid receptor genes have been generated to assess the role of nuclear receptors as transducers of the retinoid signal during vertebrate development. Mice with mutations that disrupt all isoforms of the RAR alpha, RAR beta and RAR gamma genes as well as for the individual RAR alpha 1, RAR beta 2 and RAR gamma 2 have been described. By breeding the RAR alpha 1 and RAR beta strains together we have generated double mutants which have striking phenotypes not discernible in mice homozygous for the individual mutations. Mice lacking both RAR alpha 1 and RAR beta died shortly after birth because of hypoxia, although individual RAR alpha 1 and RAR beta mutants were phenotypically normal. As previously observed in RAR compound mutants, histological examination of 18.5 dpc fetuses of RAR alpha 1 -/-beta-/- double mutants revealed a number of congenital malformations which in many respects were similar to those observed in fetuses of vitamin A-deficient mothers. The regions of congenital defects in RAR alpha 1 -/-beta-/- double mutants included the eye, the skull, the respiratory tract, the heart, the aortic arch-derived great vessels, and urogenital system. The penetrance of malformations in RAR alpha 1 -/-beta-/- mutants was greater than that in the reported RAR alpha 1 -/-beta 2-/- double mutants. Moreover, RAR alpha 1 -/-beta-/- mutants exhibited hypoplastic lungs and ossified fusion between basioccipital and exoccipital bones that were not reported in the RAR alpha 1 -/-beta2-/- animals, and displayed ectopic thymus and an unique defect in testis suggesting specific roles for RAR beta 1, 3 and/or 4 isoforms in these structures. The RAR alpha 1 single mutant animals as well as RAR alpha 1-/- beta-/- double mutant mice were susceptible to the teratogenic effects of RA, demonstrating that RAR alpha 1 and RAR beta isoforms singly or in combination do not play a major role in RA-induced craniofacial malformation and limb deformities.

Retinoic acids (RA) are vitamin A derivatives essential for normal embryonic development and viability of vertebrates. The RA signal is mediated by two distinct classes of receptors, RA receptors (RARs) and retinoid X receptors (RXRs). The RAR family is composed of three genes: RAR alpha, beta, and gamma. The expression of RAR beta gene is spatially and temporally restricted in certain structures in the developing embryo, suggesting that RAR beta could play specific roles during morphogenesis. Four isoforms of the RAR beta gene (beta 1-beta 4) are generated by differential usage of promoters and alternative splicing. It has recently been demonstrated that the RAR beta 2 isoform is dispensable for normal development. To ascertain the function of all RAR beta isoforms in vivo, we have generated a mutation that disrupts all isoforms of the RAR beta gene in the mouse by gene targeting in embryonic stem cells. Mice homozygous for the mutation are viable and fertile with no externally apparent abnormalities. During development, 1/11 RAR beta mutant embryos showed fusion of the ninth and tenth cranial ganglia on both sides of the hindbrain. However, no obvious alterations in the spatial pattern of expression of Hoxb-1, Hoxb-4 and Hoxb-5 were observed in day 9.5 p.c. embryos. The RAR beta null mutation did not alter the pattern or extent of the limb and craniofacial malformations induced by RA excess, suggesting that RAR beta may not be mandatory to mediate the observed teratological effects of RA in these structures. These experiments demonstrate that RAR beta isoforms are not absolutely required for embryonic development and provide additional support to the concept of functional redundancy among members of the RAR family.

The renin-angiotensin system (RAS) plays an important role in the development and regulation of arterial pressure during a variety of physiologic and pathophysiologic conditions. In the classic RAS, angiotensin I (Ang I) is formed by the proteolytic cleavage of the N-terminus of angiotensinogen (AGT) by renin. Angiotensin converting enzyme (ACE) in turn converts the decapeptide Ang I to the octapeptide angiotensin II (Ang II). Ang II exerts the biological functions of the RAS by binding to the two major Ang II receptors: Ang II receptor type I (AT1R) and type II (AT2R). Ang II has a similar affinity for both receptors. In addition to the presence of RAS in circulating blood, the actions of RAS can occur via generation and activity of RAS components at tissue sites. The presence of a local RAS has been demonstrated in several tissues, including human placenta.1–6 There is significant evidence to suggest that the local RAS in the placenta is involved in physiologic and pathophysiologic processes during pregnancy.7,8 Ang II is involved in the regulation of uteroplacental vascular resistance and blood flow.9 Low Ang II concentrations increase uteroplacental blood flow, whereas high concentrations of Ang II reduce blood flow.10,11 Additionally, Ang II activates plasminogen activator inhibitor-1 gene expression through AT1R and, thus, inhibits human trophoblast invasion, a process involved in the physiological union between maternal and fetal circulatory systems.12 To facilitate the research of RAS regulation in the placenta and study of the underlying mechanisms, an in vitro system, such as a placental cell line, is needed. Here we report a human placental cell line, CRL-7548, that expresses all the components of the classic RAS necessary for function.

Insulinlike growth factor binding proteins modulate the action of the insulinlike growth factors in various bioassays and may regulate the bioavailability of the insulinlike growth factors in vivo. Because the insulinlike growth factors may influence hepatic regeneration, we have examined the effect of partial hepatectomy on serum insulinlike growth factor binding proteins and on the abundance of insulinlike growth factor binding protein-1 messenger RNA in the liver. All rats were fasted before and after partial hepatectomy or sham operation to avoid the confounding effects of difference in food intake. Using a conventional protocol, 70% of the liver was removed, and groups of four or five rats were killed at different intervals after partial hepatectomy. Sham-operated rats served as controls. Pooled sera from each group of rats were analyzed by ligand blotting with 125I-insulinlike growth factor-I. Liver RNA from individual rats was analyzed by slot-blot and Northern-blot hybridization. A small decrease in the 39- to 42-kD insulinlike growth factor binding protein was apparent in sera from both the sham-operated and partial hepatectomized rats. In contrast, a dramatic increase (fivefold) in the 29-kD serum insulinlike growth factor binding protein (insulinlike growth factor binding protein-1) was apparent only in the partial hepatectomized rats. Hepatic insulinlike growth factor binding protein-1 messenger RNA abundance was significantly increased (1.99 ± 0.18-fold; p

The low mol wt insulin-like growth factor-binding protein (IGFBP-1) originally isolated from amniotic fluid has been considered to be GH independent. In this report we have examined the effect of GH on hepatic IGFBP-1 expression in the hypophysectomized rat. Using a rat IGFBP-1 cDNA probe we now demonstrated that hepatic IGFBP-1 expression is up-regulated in the GH-deficient rat. In addition, using ligand blotting, an increase in the abundance of a 30-kDa [125I]IGF-I-BP was detected in both hepatic extracts and serum from hypophysectomized rats. After a single ip injection of GH, the IGFBP-1 transcription rate was reduced within 30 min to the levels seen in the sham-operated control rats. Similarly, hepatic IGFBP-1 mRNA abundance was reduced after both acute GH injection and chronic GH treatment for 8 days. These data demonstrate that IGFBP-1 expression is inversely regulated by GH.

Multiple factors contribute to the growth retardation which is a characteristic feature of uncontrolled diabetes. In this report we have examined the effects of streptozotocin-induced (STZ) diabetes on expression of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-1 (IGFBP-1) in various tissues. As early as 7 days after STZ administration there was a modest reduction in IGF-I mRNA abundance. The reduction (10-30%) was of similar magnitude in each of the 7 tissues examined; liver, kidney, lung, diaphragm, quadraceps, heart and adipose tissue. However, the reduction achieved statistical significance only in the lung (p less than 0.05) and diaphragm (p less than 0.01). A further reduction in IGF-I mRNA abundance was seen in many tissues, 32 and 91 days after STZ administration. In contrast to the decrease in IGF-I mRNA, IGFBP-1 mRNA was significantly increased in the liver and kidney of diabetic rats. IGFBP-1 mRNA was detectable at only very low levels in other tissues but was increased in diabetic rats compared non-diabetic rats. In diabetic rats, a highly significant correlation (R = 0.75, p less than 0.001) between hepatic IGFBP-1 mRNA and glucose was observed whereas there was no significant correlation between serum glucose and hepatic IGF-I mRNA abundance (R = 0.24, p = NS). Treatment of diabetic rats with insulin resulted in a small, non significant increase in hepatic and renal IGF-I mRNA and a significant decrease in renal IGFBP-1 mRNA abundance. The observations reported here are consistent with the hypothesis that diminished IGF-I expression and inhibition of available IGF-I by increased levels of IGFBP-1 may explain the impaired growth seen in diabetic animals.

The insulin-like growth factors (IGF) are present in the serum, other biological fluids and tissue extracts in association with high affinity binding proteins1. When human or adult rat serum is analyzed by gel permeation chromatography two groups of IGF binding proteins are identified. These have apparent molecular mass of 150-200 kDa and 30–45 kDa1–3. However, more detailed analyses of sera using SDS polyacrylamide gel electrophoresis and ligand blotting with either 1251-IGF-I or 125I-IGF-II, identifies at least 5 bands. The approximate size of these binding proteins in rat serum when analyzed on reduced gels is 42, 40, 39, 30, 29 and 24 kDa 4–6. In some case where antibody probes are available it has been possible to identify the various binding proteins detected by ligand blotting however since a number of IGF binding proteins appear to be of similar molecular weight, it is possible that a band identified by ligand blotting may consist of more than one binding protein. Although the exact number of these binding proteins present in serum remains to be determined, evidence for the existence of at least five distinct binding proteins has been presented in the recent literature. Four of these binding proteins have been extensively characterized and cDNAs encoding these binding proteins have been cloned7–11.

Glucocorticoid excess is associated with growth retardation in both man and experimental animals. We have previously reported that dexamethasone (DXM) inhibits growth hormone induction of insulin-like growth factor-I (IGF-I) mRNA in the hypophysectomized rat and reduces steady-state IGF-I mRNA levels in the intact rat. However, in these experiments, DXM had surprisingly little effect on serum IGF-I concentrations. Here, we have examined the effect of DXM on hepatic insulin-like growth factor binding protein-1 (IGFBP-1) mRNA levels and on serum IGFBP-1 concentrations. After a single ip injection of DXM, 6 micrograms/100 g body wt, IGFBP-1 mRNA increased 2.24 +/- 0.25-fold, P less than 0.05 at 1 h and declined to normal levels in 6-12 h. A dose-dependent increase in increased hepatic IGFBP-1 mRNA abundance was seen in rats killed 1 h after an ip injection of DXM, 0.1 to 60 micrograms/100g body wt. As little as 1 microgram/100g body wt, significantly enhanced hepatic IGFBP-1 mRNA levels; 2.02- +/- 0.38-fold, P less than 0.05. This effect appeared to be post-transcriptional, since IGFBP-1 transcription in hepatic nuclei from rats treated with DXM was not significantly different from untreated rats. When DXM, 1 microgram/rat, was administered daily for 6 days a significant increase in IGFBP-1 mRNA was detected; 2.02 +/- 0.39-fold, P less than 0.05. A more marked increase was seen with 6 and 60 micrograms/rat of DXM; 4.47 +/- 1.08- and 10.61 +/- 0.31-fold, respectively. Serum was analyzed by sodium dodecyl sulfate-plyacrylamide gel electrophoresis and immunoblotting using antisera raised against a synthetic peptide derived from the predicted sequence of rat IGFBP-1. The antisera recognized a doublet of approximately 30 kDa. A dose-dependent increase in the abundance of these BPs were seen in the serum from rats treated chronically with DXM. The observations reported here clearly demonstrate that DXM increases hepatic IGFBP-1 mRNA and serum IGFBP-1 concentrations. If IGFBP-1 functions to decrease the bioavailability of IGF-1 in vivo the enhanced expression of IGFBP-1 may be an additional mechanism whereby glucocorticoid excess results in growth retardation.

To investigate differences in growth hormone (GH) and estrogen activation of insulin-like growth factor-I (IGF-I) expression, we have examined the effects of cycloheximide on hepatic and uterine IGF-I mRNA abundance in response to GH and 17 beta-estradiol (E2) respectively. In hypophysectomized (hypox) rats a single injection of GH significantly increased hepatic IGF-I mRNA 3.65 +/- 0.68-fold, P less than 0.005, 6 h after injection. Administration of cycloheximide 30 min prior to GH injection completely abolished this response. In contrast, in pituitary intact rats killed 6 h after administration of cycloheximide, hepatic IGF-I mRNA abundance was not significantly different from untreated control rats although the serum IGF-I concentration was significantly reduced; 119.9 +/- 11.8 vs. 270.2 +/- 48.7 ng/ml, P less than 0.005. In immature rats, injection of E2 (1 micrograms/100 g body weight) significantly increased uterine IGF-I mRNA 4.1 +/- 0.4-fold. Cycloheximide did not block the E2-induced increase in IGF-I mRNA but rather significantly enhanced the IGF-I response. These data indicate that continuing protein synthesis is required for GH induction of hepatic IGF-I mRNA in the hypox rat but is not required for E2 induction of uterine IGF-I mRNA. Furthermore, in pituitary-intact rats protein synthesis is not required for maintenance of hepatic IGF-I mRNA levels.

Exogenous glucocorticoids are potent inhibitors of skeletal growth in experimental animals and children. The mechanism whereby glucocorticoids exert this effect is unclear, and circulating somatomedin levels have been reported to be low, normal, or high in this situation. Since recent studies have emphasized the importance of autocrine or paracrine insulin-like growth factor I (IGF-I), we have examined the effects of dexamethasone (DXM) on IGF-I gene expression in the hypophysectomized (hypox) and pituitary intact rat. An increase in IGF-I messenger RNA (mRNA) abundance of approximately 5-, 3-, 2-, and 1.5-fold in the liver, proximal tibia, lung, and kidney, respectively, was seen in hypox rats killed 6 h after injection of human GH (100 micrograms/100 g body wt). As little as 1 micrograms/100 g body wt of DXM administered 3 h before GH injection significantly reduced GH induction of IGF-I mRNA in the liver and the proximal tibia (P less than 0.05 and P less than 0.005, respectively). Higher doses of DXM were required to reduce IGF-I mRNA abundance in the kidney and lung. Of the tissues examined the order of sensitivity to DXM was liver greater than tibia greater than kidney greater than lung. In contrast to the effects of DXM on tissue IGF-I mRNA abundance, an approximately 10-fold higher dose of DXM was required to inhibit the GH-induced rise in serum IGF-I concentration. The effect of DXM on steady state IGF-I mRNA abundance in pituitary-intact rats which were killed 9 h after DXM was also examined. The reduction in IGF-I mRNA abundance required higher doses of DXM (6-360 micrograms/100 g body wt) and was less marked in pituitary-intact rats than in GH-treated hypox rats. In the pituitary-intact rats the order of sensitivity to DXM was tibia greater than liver greater than lung greater than kidney. In acute studies serum IGF-I levels were not decreased by any dose of DXM; rather a significant increase in serum IGF-I was apparent in pituitary intact rats, treated with the lowest and highest doses of DXM. When DXM was administered daily for 6 days to pituitary intact rats, body wt gain and hepatic and tibial IGF-I mRNA abundance were significantly reduced. No significant changes were seen in serum IGF-I concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)