Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells

We aimed to determine whether three-dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n=4) harvested at 80-120 days of gestation by low-density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n=28) were maintained in a rotating bioreactor with serum-free medium supplemented with transforming growth factor-beta1 for 4-12 weeks. Similar constructs seeded with fetal chondrocytes (n=13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n=10). Statistical analysis was by analysis of variance and Student's t-test (p<.01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix-specific staining. In the CB constructs, there was a significant time-dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type-II collagen (C-II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C-II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C-II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.     

Role of C-C chemokine receptors 1 and 5 and CCL3/macrophage inflammatory protein-1alpha in the cutaneous Arthus reaction: possible attenuation of their inhibitory effects by compensatory chemokine production

The deposition of immune complexes induces an acute inflammatory response with tissue injury. Immune complex-induced tissue injury is mediated by inflammatory cell infiltration that is highly regulated by multiple chemokines. To assess the role of the chemokine receptors CCR1 and CCR5, and a ligand for these receptors CCL3/macrophage inflammatory protein-1alpha, in this pathogenic process, the reverse passive cutaneous Arthus reaction was induced in mice lacking CCR1, CCR5, or CCL3. Edema was significantly attenuated in CCR1-deficient (CCR1(-/-)) and CCL3(-/-) mice but not CCR5(-/-) mice, compared with wild-type mice. Numbers of infiltrating neutrophils and mast cells were reduced in CCL3(-/-) and CCR1(-/-) mice, respectively, compared with wild-type mice. CCR1 and CCR5 were expressed on neutrophils and mast cells. Remarkably, the intradermal mRNA expression of CCL5/RANTES, another ligand for CCR1 and CCR5, was increased in CCR5(-/-) and CCL3(-/-) mice, compared with wild-type mice, while the cutaneous CCL3 mRNA expression was augmented in CCR1(-/-) and CCR5(-/-) mice. These results indicate that CCR1, CCR5, and CCL3 cooperatively contribute to the cutaneous Arthus reaction, and also suggest that enhanced expression of CCL3 and CCL5 compensates for the loss of CCR1, CCR5, and CCL3 in the reaction.     

BAG1 over-expression in brain protects against stroke

The co-chaperone BAG1 binds and regulates 70 kDa heat shock proteins (Hsp70/Hsc70) and exhibits cytoprotective activity in cell culture models. Recently, we observed that BAG1 expression is induced during neuronal differentiation in the developing brain. However, the in vivo effects of BAG1 during development and after maturation of the central nervous system have never been examined. We generated transgenic mice over-expressing BAG1 in neurons. While brain development was essentially normal, cultured cortical neurons from transgenic animals exhibited resistance to glutamate-induced, apoptotic neuronal death. Moreover, in an in vivo stroke model involving transient middle cerebral artery occlusion, BAG1 transgenic mice demonstrated decreased mortality and substantially reduced infarct volumes compared to wild-type littermates. Interestingly, brain tissue from BAG1 transgenic mice contained higher levels of neuroprotective Hsp70/Hsc70 protein but not mRNA, suggesting a potential mechanism whereby BAG1 exerts its anti-apoptotic effects. In summary, BAG1 displays potent neuroprotective activity in vivo against stroke, and therefore represents an interesting target for developing new therapeutic strategies including gene therapy and small-molecule drugs for reducing brain injury during cerebral ischemia and neurodegenerative diseases.     

The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture

Our research group aims to develop an osteochondral composite using type II collagen gel with hydroxyapatite (HAp) deposited on one side. Soaking gels in Ca2+ and phosphate solution is indispensable to HAp deposition, so relationships between cell behavior and Ca2+ concentration were examined in two- and three-dimensional cultures. The present results indicate that 2-4 mM Ca2+ is suitable for proliferation and survival of osteoblasts, whereas slightly higher concentrations (6-8 mM) favor osteoblast differentiation and matrix mineralization in both 2- and 3-dimensional cultures. Higher concentrations (>10 mM) are cytotoxic. Purely from the perspective of calcium deposition, higher concentrations lead to increased accumulation of Ca2+. Culturing cells in phosphate-containing gel in media with Ca2+ also leads to time-dependent formation of HAp in the gel. Considering the viability of embedded cells, culturing scaffolds in media with Ca2+ concentrations around 5mM is useful for both HAp deposition and osteoblast behavior.     

Otx1 function overlaps with Otx2 in development of mouse forebrain and midbrain

Background: We previously reported that the homozygous mutation of Otx2 gene, a mouse cognate of the Drosophila head gap gene orthodenticle , causes failure in the development of the rostral head anterior to rhombomere 3, which may correspond to earlier Otx2 expression in cells destined for the anterior mesoendoderm. At the same time, the Otx2 heterozygous mutation displayed a phenotype characterized as otocephaly, probably related to expression in the anterior neuroectoderm at the subsequent pharyngula stage. Defects were characteristic in the most anterior and posterior regions of Otx2 expression where Otx1 , another mouse cognate of orthodenticle , is not or weakly expressed. They were not found in the region where Otx1 is expressed.
Results: In the present work, Otx1 null mutant mice were generated by gene targeting in embryonic stem cells. No defects were apparent in the regionalization of the early embryonic rostral brain. The newborn brain defects were subtle and most likely related to later Otx1 -unique expression. Otx1 and Otx2 double heterozygous mutant brains, however, exhibited marked defects throughout the fore- and midbrains, where defects were not apparent with a single mutation alone.
Conclusions: Otx1 and Otx2 play synergistic roles in the development of the forebrain and midbrain where both genes are expressed.     

Immunohistochemistry and angiography in adenomatous hyperplasia and small hepatocellular carcinomas

The sinusoidal structure and blood supply of 38 liver nodules less than 2 cm In diameter were Investigated. There were 18 cases of adenomatous hyperplasia (AH) and 20 cases of hepatocetlular carcinoma (HCC). Growth pattern, encapsulation and vascularity were examined, and Immunohistochemistry performed for factor VIII related antigen (factor VIII), type IV collagen (collagen IV), lamlnln and CD68. There were significant differences between AH and small HCC, except for the expression of CD68. There were differences In tumor size, vasculartty and the components of the basement membrane between AH and small, well differentiated HCC. The cases of AH were supplied by the portal system and maintained the sinusoidal structure, but small well-differentiated HCC were supplied by a mixture of portal and arterial vessels. In spite of their small size, moderately and poorly differentiated HCC had capillary and were supplied by branches of the hepatic artery.     

Total energy expenditure of elite synchronized swimmers measured by the doubly labeled water method

To determine the daily energy requirement of elite synchronized swimmers during moderate-intensity training, the average daily energy expenditure measured by the doubly labeled water method, was calculated for nine female Japanese national team synchronized swimmers [four senior; mean (SD) 22.5 (1.0) years old, 52.2 (3.6) kg, and five junior; 17.6 (1.1) years old, 52.8 (2.3) kg]. Their total energy expenditure (TEE) was 11.5 (2.8) MJ · day⁻¹ [2738 (672) kcal · day⁻¹]. When compared with estimated energy requirements derived from “Recommended Dietary Allowances for the Japanese”, 12.1 (0.6) MJ · day⁻¹ [2897 (139) kcal · day⁻¹], there was no difference between mean actual and estimated energy requirements. However, there were considerable differences observed on an individual basis. Their energy intake, estimated from 7- day self-reported dietary records, was 8.9 (1.7) MJ · day⁻¹ [2128 (395) kcal · day⁻¹], which was significantly lower than their TEE (P < 0.05). Resting energy expenditure (REE), as determined by indirect calorimetry, was 5.2 (0.3) MJ · day⁻¹ [1247 (75) kcal · day⁻¹]. Their physical activity level (TEE/REE) was 2.18 (0.43). These results demonstrate that the TEE values of elite female synchronized swimmers are not dissimilar to those reported for athletes participating in other sports, especially competitive swimmers during moderate-intensity training. Accepted: 26 May 2000