A Concise Review on the Use of Mesenchymal Stem Cells in Cell Sheet-Based Tissue Engineering with Special Emphasis on Bone Tissue Regeneration.

The integration of stem cell technology and cell sheet engineering improved the potential use of cell sheet products in regenerative medicine. This review will discuss the use of mesenchymal stem cells (MSCs) in cell sheet-based tissue engineering. Besides their adhesiveness to plastic surfaces and their extensive differentiation potential in vitro, MSCs are easily accessible, expandable in vitro with acceptable genomic stability, and few ethical issues. With all these advantages, they are extremely well suited for cell sheet-based tissue engineering. This review will focus on the use of MSC sheets in osteogenic tissue engineering. Potential application techniques with or without scaffolds and/or grafts will be discussed. Finally, the importance of osteogenic induction of these MSC sheets in orthopaedic applications will be demonstrated.

Plasma Amino-Terminal Propeptide of C-Type Natriuretic Peptide Concentration in Normal-Weight and Obese Children.

OBJECTIVE: In studies on the relationship between amino-terminal propeptide of C-type natriuretic peptide (NT-proCNP) concentration and height velocity in children, CNP has been implicated as an emerging new growth marker during childhood. It has been reported that besides its well-studied role in growth, plasma CNP levels are reduced in overweight and/or obese adolescents, suggesting CNP as a potential biomarker in childhood obesity. The primary goal of this study was to test this hypothesis in a Turkish population. METHODS: Consent was taken from 317 children [ages 0-18 (158 girls, 159 boys)] and their parents. All subjects were physically examined; anthropometric measurements were obtained. Body mass index was calculated. During routine blood work, 1 mL extra blood was taken. Plasma NT-proCNP concentration was measured by enzyme-linked immunosorbent assay. RESULTS: Results confirmed the previously described relationship between plasma NT-proCNP concentration and growth velocity. Plasma NT-proCNP concentration showed a negative correlation with age, weight, and height in children. Gender was not a factor that alters the age-dependent plasma NT-proCNP concentration until puberty. CONCLUSION: Unlike previous reports, plasma NT-proCNP concentration of overweight/obese children was not significantly lower than that of children with normal weight in age groups analyzed in a Turkish population. Thus, it is too early to conclude that CNP is a potential biomarker in childhood obesity. Further studies are necessary to address this question.

Matrilin-3 as a putative effector of C-type natriuretic peptide signaling during TGF-ß induced chondrogenic differentiation of mesenchymal stem cells.

C-type natriuretic peptide (CNP) signaling has been implicated as an important regulator of chondrogenic differentiation during endochondral bone development. This preliminary study further investigated the putative effectors and/or targets of CNP signaling in transforming growth factor (TGF)-ß induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs). Previously characterized human trabecular bone derived MSCs were induced either with only TGF-ß1 or with a combination of TGF-ß1 and CNP in micromass culture for 10 or 20 days. Genome wide gene expression profile changes in between these two groups were analyzed on day-10 or day-20 of culture. Results revealed that there were only 7 genes, whose expression change was fourfolds or higher in TGF-ß1 and CNP fed group in comparison to only TGF-ß1 fed group. The up-regulated genes included matrilin-3 (MATN3), engulfment and cell motility 1 (ELMO1), CD24, and DCN1, defective in cullin neddylation 1, domain containing 1 (DCUN1D1). The down-regulated genes, on the other hand, included LIM domain kinase 2 (LIMK2), Ewing sarcoma breakpoint region 1, and guanine nucleotide binding protein (G protein), gamma 12 (GNG12). The up-regulation of MATN3 was confirmed on the basis of RT-PCR. The known literature on both CNP signaling and MATN3 function in chondrogenesis match with each other and suggest MATN3 as a putative effector and/or target of CNP signaling during this process.

Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-ß1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells.

This study investigated the involvement of CNP-3, chick homologue for human C-type natriuretic peptide (CNP), in TGF-ß1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells (MSCs). Chondrogenic differentiation of MSCs in pellet cultures was induced by TGF-ß1. Chondrogenic differentiation and glycosaminoglycan synthesis were analyzed on the basis of basic histology, collagen type II expression, and Alcian blue staining. Antibodies against CNP and NPR-B were used to block their function during these processes. Results revealed that expression of CNP-3 and NPR-B in MSCs were regulated by TGF-ß1 in monolayer cultures at mRNA level. In pellet cultures of MSCs, TGF-ß1 successfully induced chondrogenic differentiation and glycosaminoglycan synthesis. Addition of CNP into the TGF-ß1 supplemented chondrogenic differentiation medium further induced the glycosaminoglycan synthesis and hypertrophy of differentiated chondrocytes in these pellets. Pellets induced with TGF-ß1 and treated with antibodies against CNP and NPR-B, did show collagen type II expression, however, Alcian blue staining showing glycosaminoglycan synthesis was significantly suppressed. In conclusion, CNP-3/NPR-B signaling may strongly be involved in synthesis of glycosaminoglycans of the chondrogenic matrix and hypertrophy of differentiated chondrocytes during TGF-ß1 induced chondrogenic differentiation of MSCs.

Dose dependent effect of C-type natriuretic peptide signaling in glycosaminoglycan synthesis during TGF-ß1 induced chondrogenic differentiation of mesenchymal stem cells.

Recent investigations credited important roles to C-type natriuretic peptide (CNP) signaling during chondrogenesis. This study investigated the putative role of CNP in transforming growth factor (TGF)-ß1 induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs) in pellet culture. MSCs were derived from human trabecular bone and were characterized on the basis of their cell surface antigens and adipogenic, osteogenic, and chondrogenic differentiation potential. TGF-ß1 induced chondrogenic differentiation and glycosaminoglycan (GAG) synthesis was analyzed on the basis of basic histology, collagen type II, Sox 9 and aggrecan expressions, and Alcian blue staining. Results revealed that human trabecular bone-derived MSCs express CNP and NPR-B analyzed on the basis of RT-PCR and immunohistochemistry. In pellet cultures of MSCs TGF-ß1 successfully induced chondrogenic differentiation and GAG synthesis. RT-PCR analyses of both CNP and NPR-B during this process revealed an activation of this signaling pathway in response to TGF-ß1. Similar cultures induced with TGF-ß1 and treated with different doses of CNP showed that CNP supplementation at 10(-8) and 10(-7) M concentrations significantly increased GAG synthesis in a dose dependent manner, whereas at 10(-6) M concentration this stimulatory effect was diminished. In conclusion, CNP/NPR-B signaling pathway is activated during TGF-ß1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may strongly be involved in GAG synthesis during this process. This effect is likely to be a dose-dependent effect.

Immunohistochemical demonstration of nerve endings in iliolumbar ligament.

STUDY DESIGN: Immunohistochemical study on fresh cadaver specimens. OBJECTIVE: Assessment of mechanoreceptor and nociceptor levels and distribution in iliolumbar ligament. SUMMARY AND BACKGROUND DATA: The function of iliolumbar ligament and its role in low back pain has not been yet fully clarified. Understanding the innervation of this ligament should provide a ground which enables formation of stronger hypotheses. METHODS: Bilateral 30 iliolumbar ligaments of 15 fresh cadavers were included in the study. Morphologic properties were recorded and the ligaments were examined by focusing on 3 main parts: ligament, bone insertions, and tendon body. Assessment of mechanoreceptor and nociceptor levels and their distribution in iliolumbar ligament were performed on the basis of immunohistochemistry using the S-100 antibody specific for nerve tissue. RESULTS: Iliac wing insertion was found to be the richest region of the ligament in terms of mechanoreceptors and nociceptors. Pacinian (type II) mechanoreceptor was determined to be the most common (66.67%) receptor followed by Ruffini (type I) (19.67%) mechanoreceptor, whereas free nerve endings (type IV) and Golgi tendon organs (type III) were found to be less common, 10.83% and 2.83%, respectively. CONCLUSION: Immunohistochemical staining has shown that iliolumbar ligament had a rich nerve tissue. Those results indicate that ILL plays an important role in proprioceptive coordination of lumbosacral region alongside its known biomechanic support function. Moreover, the presence of type IV nerve endings suggest that the injury of this ligament might contribute to the low back pain.

The effects of copper sulfate on liver histology and biochemical parameters of term Ross broiler chicks.

Copper is an essential trace element that is extremely toxic to organisms and organs at high doses. We have investigated the histological and biochemical effects of a toxic dose of copper sulfate on the liver of term Ross broiler chicks. Fertilized eggs were divided into three groups: experimental, injected with 50 mcg/0.1 ml copper sulfate in the air chambers on day 1; sham, injected with 0.1 ml saline; and control, no injection. Term chicks were killed and their livers investigated histologically, with hematoxylin-eosin-stained sections examined under light microscopy, and biochemically, for malondialdehyde and glutathione levels. Histological examinations showed copper-treated samples with granular degeneration and necrosis of hepatocytes and impairment to the cell lining of the remark cords. The samples had a congestive appearance, with blood in the vena centralis and sinusoids, slight connective tissue increase, and lymphocyte infiltration. Control and sham group sections had normal appearances. As oxidative damage parameters, in the copper-treated group, malondialdehyde levels were increased and glutathione levels decreased. In the sham and control groups, there were no significant differences. At this toxic dose, copper sulfate shows oxidative damage according to the histology of term chick liver that are confirmed biochemically by the changes in malondialdehyde and glutathione levels.

Effects of different corticosteroids on the brain weight and hippocampal neuronal loss in rats.

Equivalent antiinflammatory doses of steroids including betamethasone, methylprednisolone and dexamethasone were administered in the neonatal period in a rat model. In situ cell death in hippocampus quantified by Terminal Deoxynucleated Transferase Nick-End Labeling and on ratio of brain to body weight was investigated. Apoptotic index (AI) was significantly higher in methylprednisolone, and high dose dexamethasone groups than the other groups. AI in "Cornu ammonis 1" (CA1) and "Cornu ammonis 3" (CA3) subregions of high dose dexamethasone group was the highest among the five groups tested. AI in CA3 subregions of methylprednisolone group was also significantly higher than the control, betamethasone and low dose dexamethasone groups. AI in CA1 subregion were not different among control, betamethasone, methylprednisolone and low dose dexamethasone groups. In addition, high dose dexamethasone resulted significant decrease in the ratio of brain weight to body weight in comparison to all other groups tested. In conclusion, betamethasone and low dose dexamethasone may be better alternative treatments among agents tested in this study for chronic lung disease (CLD).

Penicillin-induced epilepsy model in rats: dose-dependant effect on hippocampal volume and neuron number.

This study was designed to evaluate the penicillin-induced epilepsy model in terms of dose-response relationship of penicillin used to induce epilepsy seizure on hippocampal neuron number and hippocampal volume in Sprague-Dawley rats. Seizures were induced with 300, 500, 1500 and 2000IU of penicillin-G injected intracortically in rats divided in four experimental groups, respectively. Control group was injected intracortically with saline. Animals were decapitated on day 7 of treatment and brains were removed. The total neuron number of pyramidal cell layer from rat hippocampus was estimated using the optical fractionator method. The volume of same hippocampal areas was estimated using the Cavalieri method. Dose-dependent decrease in hippocampal neuron number was observed in three experimental groups (300, 500 and 1500IU of penicillin-G), and the effects were statistically significant when compared to the control group (P<0.009). Dose-dependent decrease in hippocampal volume, on the other hand, was observed in all three of these groups; however, the difference compared to the control group was only statistically significant in 1500IU of penicillin-G injected group (P<0.009). At the dose of 2000IU penicillin-G, all animals died due to status seizures. These results suggest that the appropriate dose of penicillin has to be selected for a given experimental epilepsy study in order to demonstrate the relevant epileptic seizure and its effects. Intracortical 1500IU penicillin-induced epilepsy model may be a good choice to practice studies that investigate neuroprotective mechanisms of the anti-epileptic drugs.

Increased tunel positive cells in CA1, CA2, and CA3 subfields of rat hippocampus due to copper and ethanol co-exposure.

Copper (Cu) is an essential element for life. However, it is toxic at excessive doses, whereas exposure to ethanol (EtOH) has known to cause morphological changes, degeneration, and neuronal loss in central nervous system. A previous investigation by the authors' group showed that Cu and EtOH co-treatment cause severe hippocampal neuronal loss in CA1, CA2, and CA3 subfields of rat hippocampus. This study was designed to analyze the possible mechanism(s) of action of this effect. In addition, the possible neurogenesis in response to a potent neurodegenerative treatment in rat hippocampus was analyzed. Results demonstrated that Cu and EtOH induced neuronal loss in rat hippocampus was in correlation with the increased cell death analyzed on the basis of TdT-mediated dUTP nick end labeling (TUNEL) assay. On the other hand, neuronal regenerative activity was detectable in analyzed CA1, CA2, and CA3 subfields of the rat hippocampus analyzed on the basis of 5-bromo-2'-deoxy-uridine (BrdU) labeling assay; however, this activity in treated group was not significantly different from that of control group.

C-type natriuretic peptide regulation of limb mesenchymal chondrogenesis is accompanied by altered N-cadherin and collagen type X-related functions.

AMDM, a form of osteochondrodysplasia, is due to the loss-of-function mutations in NPR-B gene. This study investigated the functional involvement of CNP-3, chick homolog of human CNP, and its receptor NPR-B in chondrogenesis utilizing the micromass culture of the chick limb mesenchymal cells. Results revealed CNP-3 and NPR-B expression in the chick limb bud making stage-specific peak levels first at Hamburger-Hamilton stage 23-24, and second at stage 30-31, corresponding to pre-chondrogenic mesenchymal condensation and initiation of chondrogenic maturation-hypertrophy in vivo, respectively. CNP-3 and NPR-B expression in vitro increased parallel to collagen type X expression, but not to that of collagen type II. Treatment of cultures with CNP significantly increased N-cadherin, and collagen type X expression, glycosaminoglycan synthesis and chondrogenesis. Collagen type II expression was not significantly affected. Thus, results implicated CNP-3/NPR-B signaling in pre-chondrogenic mesenchymal condensation, glycosaminoglycan synthesis and late differentiation of chondrocytes in the process of endochondral ossification.

Apoptotic cell death and its relationship to gastric carcinogenesis.

AIM: To investigate the apoptotic process of cells within the intestinal metaplasia areas co-localizing with chronic gastritis and gastric carcinomas and to analyze the involvement of proteins regulating apoptosis in the process of intestinal metaplasia related gastric carcinogenesis. METHODS: Forty-two gastric carcinoma and seventeen chronic gastritis cases were included in this study. All cases were examined for the existence of intestinal metaplasia. Ten cases randomly selected from each group were processed for TUNEL assay. TUNEL positive cells within the intestinal metaplasia areas, co-localizing either to gastric carcinoma or chronic gastritis, were counted and converted to apoptotic indices. In addition, p53, bcl-2 and bax expression patterns within these tissues were analyzed on the basis of immunohistochemistry. RESULTS: Twenty-eight of the cases were intestinal and 14 of the cases were diffuse type adenocarcinomas. 64% (27/42) of the gastric carcinoma cases had intestinal metaplasia. Intestinal metaplasia co-localized more with intestinal type carcinomas compared with diffuse type carcinomas [75% (21/28) vs 42% (6/14), respectively; P

Serum or plasma cartilage oligomeric matrix protein concentration as a diagnostic marker in pseudoachondroplasia: differential diagnosis of a family.

Pseudoachondroplasia (PSACH) is an autosomal-dominant osteochondrodysplasia due to mutations in the gene encoding cartilage oligomeric matrix protein (COMP). Clinical diagnosis of PSACH is based primarily on family history, physical examination, and radiographic evaluation, and is sometimes extremely difficult, particularly in adult patients. Genetic diagnosis based on DNA sequencing, on the other hand, can be expensive, time-consuming, and intensive because COMP mutations may be scattered throughout the gene. However, there is evidence that decreased plasma COMP concentration may serve as a diagnostic marker in PSACH, particularly in adult patients. Here, we report the serum and/or plasma COMP concentration-based differential diagnosis of a family with affected adult members. The mean serum and/or plasma COMP concentrations of the three affected family members alive (0.69+/-0.15 and/or 0.81+/-0.08 microg/ml, respectively) were significantly lower than those of an age-compatible control group of 21 adults (1.52+/-0.37 and/or 1.37+/-0.36 microg/ml, respectively; P<0.0001). Bidirectional fluorescent DNA sequencing-based genetic diagnosis of these patients revealed a heterozygous mutation for the nucleotide change 1532A>G in exon 14 of the COMP gene, resulting in a substitution of amino acid 511 from aspartic acid to glycine in COMP. Thus, serum and/or plasma COMP concentration may be suggested as an additional diagnostic marker to aid clinical and radiographic findings in suspected cases of PSACH.

The effect of in ovo ethanol exposure on retina and optic nerve in a chick embryo model system.

Ocular anomalies seen in children with fetal alcohol syndrome (FAS) suggest that ocular structures are sensitive to alcohol exposure during their development. This study was designed to investigate the effect of in ovo ethanol (EtOH) exposure on retinal development and myelinization of optic nerve fibers at an ultra structural level in a chick embryo model system. Prior to incubation, fertilized chicken eggs were injected once with 100 microl of either 0.9% NaCl (vehicle control), or EtOH solutions at different doses (10, 30, or 50%, v:v in 0.9% NaCl) into their air sacs and incubated at 37.5 degrees C and saturation humidity. On day 20 embryos were analyzed in terms of their viability and growth and the optic cups including the optic nerves were dissected out. Specimens were processed for electron microscopy (EM). Results showed that, EtOH significantly decreased the viability of chick embryos (P < 0.045), and caused significant prenatal growth retardation (P < 0.004) in a dose-dependant manner. Light microscopy of semi thin sections revealed that prenatal exposure to EtOH resulted in both retinal degeneration and optic nerve hypoplasia (P < 0.001) in a dose-dependant manner. EM revealed that a dose-dependant decrease in the number of myelinated nerve fibers was profound in groups exposed to EtOH (P < 0.001). Furthermore, the myelin coats observed were thinner than those seen in control embryos. In groups exposed to EtOH myelin sheets were unorganized and contained vacuolar structures in between them. The tissue in between the cells and optic nerve fibers, on the other hand, lost its intact appearance with vacuolar and vesicular structures in between them. In addition, the optic nerve fibers contained granular accumulations in EtOH exposed groups. A dose dependent degeneration was also observed in retinas of EtOH exposed groups. The effect of EtOH was profound in pigment epithelium (PE), inner plexiform layer (IPL), and ganglion cell layer (GC). Mitochondrial deficiencies, and alterations in melanin granule number and distribution dominated the defects seen in PE. On the other hand, EM findings of all the affected layers were suggestive of induced cell death in EtOH exposed groups. Thus, this study suggests retinal development with the emphasis on melanin pigmentation in PE and optic nerve myelinization as potential targets of prenatal EtOH exposure and discusses potential mechanisms of EtOH action on these tissues.

Accurate diagnosis of a homozygous G1138A mutation in the fibroblast growth factor receptor 3 gene responsible for achondroplasia.

Achondroplasia is the most common genetic form of dwarfism inherited as an autosomal dominant disorder. Individuals affected with achondroplasia have impaired ability to form bone from cartilage (endochondral bone formation). Homozygous achondroplasia is a neonatal lethal condition. The vast majority of patients with achondroplasia have a G-to-A transition at position 1138 of the fibroblast growth factor receptor 3 (FGFR3) cDNA sequence, resulting in the Gly-to-Arg substitution at position 380 of the FGFR3 protein. This mutation has been diagnosed by SfcI digestion of amplified genomic DNA. However, it has also been demonstrated that the SfcI digestion protocol does not consistently distinguish between DNA samples heterozygous and homozygous for the G1138A substitution. This study was designed to improve the molecular diagnosis based on the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques for the FGFR3 G1138A mutation. The newly designed forward primer contains one mismatch (G at position 1136) from the FGFR3 cDNA sequence (A at position 1136), thereby creating a PstI site (CTGCAG at positions 1134 to 1139) in the amplified DNA from alleles containing the G1138A mutation. The PCR-RFLP technique based on the PstI digestion of amplified genomic DNA with a novel forward primer shows 100% accuracy in diagnosis of the G1138A mutation in heterozygous and homozygous individuals.

A case with proximal femoral focal deficiency (PFFD) and fibular A/hypoplasia (FA/H) associated with urogenital anomalies.

Malformations of the lower limbs are rare and heterogeneous anomalies. Some congenital anomalies involving face, gastrointestinal system, skeletal system, urogenital system, heart, lung and diaphragma associated with lower limb malformations have been described in the literature. Here, we report a case of left proximal femoral focal deficiency (PFFD) together with fibular aplasia associated with left undescended testis and hypospadias. The putative embryologic mechanisms of lower limb defects and their possible association with lower urogenital tract malformations are also discussed.

The chick embryo chorioallantoic membrane as a model system for the study of tumor angiogenesis, invasion and development of anti-angiogenic agents.

Angiogenesis, the formation of new blood vessels, is essential for tumor growth, progression and metastasis. The development of agents that target tumor vasculature is ultimately dependent on the availability of appropriate preclinical screening assays. The chorioallantoic membrane (CAM) assay is well established and widely used as a model to examine angiogenesis, and anti-angiogenesis. This review 1) summarizes the currently used angiogenesis assays and the importance of CAM model among them; 2) summarizes the current knowledge about the development and structure of the CAM's capillary bed; 3) reports findings regarding the role played by molecular signaling pathways in angiogenesis process; 4) discusses the use, advantages and limitations of the CAM as a model for studying tumor angiogenesis and invasiveness, as well as development of angiogenic and/or anti-angiogenic agents; 5) discusses the importance of standardization of the major methodologies for all aspects of the use of the CAM in angiogenesis-related studies; 6) and finally, summarizes major findings regarding the agents developed by the use of CAM model in the study of tumor angiogenesis, invasion and development of anti-angiogenic agents.

No association of the CAG repeat length in exon 1 of the androgen receptor gene with idiopathic infertility in Turkish men: implications and literature review.

While the correlation between the CAG repeat length of the androgen receptor (AR) gene and idiopathic male infertility is still unclear, ethnic background of the population studied may play an important role in this association. The objective of this study was to determine whether changes in the CAG repeat length are associated with spermatogenic defects in Turkish infertile men. Reproductive hormone concentrations and the CAG repeat length in exon 1 of the AR gene in 47 idiopathic infertile men and 32 fertile controls were analyzed. The mean serum luteinising hormone (LH) and follicle stimulating hormone (FSH) levels were significantly higher in the infertile group than those of the control group (p < 0.0001 for both comparisons), whereas the mean serum testosterone level in the infertile group did not differ significantly from that in the control group (p = 0.16). The mean CAG repeat length of the AR gene in the infertile group did not differ significantly from that in the control group (22.28 +/- 0.37 vs 22.41 +/- 0.54, respectively; p = 0.84). In addition, the frequency of having a CAG repeat number (> or = 24) was also comparable between the infertile patients and fertile controls (31.9% vs 40.6%, respectively; p = 0.21). In conclusion, reproductive hormones with elevated LH and FSH, and normal or low testosterone levels were suggestive of partial impairment of testicular function. However, no statistically significant relationship between the length of the CAG repeat and idiopathic impaired sperm production was observed in the Turkish population studied. These results support the findings of previously published European studies, but are contrary to the findings from Caucasian and North American population studies. Thus, ethnicity and genetic backgrounds seem to be important in this association, and studies from a variety of different ethnic and genetic backgrounds using comparable patient subgroups are valuable to further evaluate this association.

Granule cell apoptosis induced by overdose copper and ethanol is counterbalanced by co-induced cellular proliferation in rat dentate gyrus.

Copper (Cu) is an essential element for life, however, is toxic at excessive doses, whereas exposure to ethanol (EtOH) has been known to cause morphological changes, degeneration and neuronal loss in central nervous system (CNS). In this study, the effect of overdose co-exposure to Cu and EtOH on dentate gyrus was investigated in rats. Analysis of apoptotic cell death on the basis of TdT-mediated dUTP nick end labeling (TUNEL) assay revealed that the rate of apoptosis was increased by 1.84 folds in treated group in comparison to that in controls (p < 0.0001). Analysis of cell proliferation on the basis of 5-bromo-2'-deoxy-uridine labeling assay, on the other hand, revealed a 1.49 fold increase in treated group when compared to controls (p < 0.006). Total number of granule cells in dentate gyrus of each group was estimated using the optical fractionator method. The results showed that mean granule cell number in dentate gyrus was 4.64% lower in treated group than that in control group, but this difference was not statistically significant (p > 0.05). These results suggest that the apoptotic effect of overdose Cu and EtOH on granule cells of dentate gyrus may be counterbalanced by the co-induced cellular proliferation, thereby maintaining the total granule cell number unaltered.

Long-term in vitro analysis of limb cartilage development: involvement of Wnt signaling.

Endochondral skeletal development involves the condensation of mesenchymal cells, their differentiation into chondrocytes, followed by chondrocyte maturation, hypertrophy, and matrix mineralization, and replacement by osteoblasts. The Wnt family of secreted proteins have been shown to play important roles in vertebrate limb formation. To examine the role(s) of Wnt members and their transmembrane-spanning receptor(s), Frizzled (fz), we retrovirally misexpressed Wnt-5a, Wnt-7a, chicken frizzled-1 (Chfz-1), and frizzled-7 (Chfz-7) in long-term (21 day) high density, micromass cultures of stage 23/24 chick embryonic limb mesenchyme. This culture system recapitulates in vitro the entire differentiation (days 1-10), growth (days 5-12), and maturation and hypertrophy (from day 12 on) program of cartilage development. Wnt-7a misexpression severely inhibited chondrogenesis from day 7 onward. Wnt-5a misexpression resulted in a poor hypertrophic phenotype by day 14. Chfz-7 misexpression caused a slight delay of chondrocyte maturation based on histology, whereas Chfz-1 misexpression did not affect the chondrogenic phenotype. Misexpression of all Wnt members decreased collagen type X expression and alkaline phosphatase activity at day 21. Our findings implicate functional role(s) for Wnt signaling throughout embryonic cartilage development, and show the utility of the long-term in vitro limb mesenchyme culture system for such studies.