Saul F. Juliao

Comparison of fresh osteochondral autografts and allografts: a canine model.

Background Osteochondral autografts and allografts have been widely used in the treatment of isolated grade IV articular cartilage lesions of the knee. However, the authors are not aware of any study that has prospectively compared fresh osteochondral autografts to fresh allografts with regard to imaging, biomechanical testing, and histology. Hypothesis The imaging, biomechanical properties, and histologic appearance of fresh osteochondral autograft and fresh allo-graft are similar with respect to bony incorporation into host bone, articular cartilage composition, and biomechanical properties. Study Design Controlled laboratory study. Methods Eighteen adult dogs underwent bilateral knee osteochondral graft implantation after creation of an Outerbridge grade IV cartilage defect. One knee received an autograft, and the contralateral knee received a fresh allograft. Nine dogs were sacrificed at 3 months, and 9 dogs were sacrificed at 6 months. Graft analysis included gross examination, radiographs, magnetic resonance imaging, biomechanical testing, and histology. Results Magnetic resonance imaging demonstrated excellent bony incorporation of both autografts and allografts. Biomechanical testing demonstrated no significant difference between autografts versus allografts versus control at 3 or 6 months (P= .36-.91). A post hoc calculation showed 80% power to detect a 30% difference between allograft and control. Histologic examination showed normal cartilage structure for both autografts and allografts. Conclusion Fresh osteochondral autograft and fresh allograft tissues are not statistically different with respect to bony incorporation, articular cartilage composition, or biomechanical properties up to 6 months after implantation. Clinical Relevance The use of fresh allograft tissue to treat osteochondral defects eliminates morbidity associated with harvesting autograft tissue without compromising the results of the surgical procedure.

Telomerase activity and oncogenesis in giant cell tumor of bone

Background. Benign giant cell tumor of bone (GCT) is a primary skeletal neoplasm with an unpredictable pattern of biologic aggressiveness and cytogenetic findings characterized by telomeric associations and telomeric reduction. The role of maintaining telomeric integrity is performed by telomerase. To determine if telomerase activity is present, cell extracts from fibroblasts and tumor cells from five patients with GCT were analyzed and compared with HeLa (a positive control cell line).

Ascorbic acid uptake and regulation of type I collagen synthesis in cultured vascular smooth muscle cells.

Background/Aims: Vascular smooth muscle cells contribute both to the structure and function of arteries, but are also involved in pathologic changes that accompany inflammatory diseases such as atherosclerosis. Since inflammation is associated with oxidant stress, we examined the uptake and cellular effects of the antioxidant vitamin ascorbic acid in cultured A10 vascular smooth muscle cells. Methods/Results: A10 cells concentrated ascorbate against a gradient in a sodium-dependent manner, most likely on the sodium-dependent vitamin C transporter type 2 (SVCT2) ascorbate transporter, which was present in immunoblots of cell extracts. Although ascorbate did not affect A10 cell proliferation, it stimulated radiolabeled proline incorporation and type I collagen synthesis. The latter was evident in the cells as increases in proα1(I) collagen and conversion of proα1(I) and proα2(I) collagen to mature forms that were released from the cells and deposited as extracellular matrix. Intracellular type I procollagen maturation was optimal at intracellular ascorbate concentrations of 200 μM and below, which were readily achieved by culture of the cells at plasma physiologic ascorbate concentrations. Conclusion: These results show that the SVCT2 facilitates ascorbate uptake by vascular smooth muscle cells, which in turn increases both the synthesis and maturation of type I collagen.

Erg is a crucial regulator of endocardial-mesenchymal transformation during cardiac valve morphogenesis

During murine embryogenesis, the Ets factor Erg is highly expressed in endothelial cells of the developing vasculature and in articular chondrocytes of developing bone. We identified seven isoforms for the mouse Erg gene. Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes. The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells. Homozygous ErgΔEx3/ΔEx3 knockout mice are viable, fertile and do not display any overt phenotype. By contrast, homozygous ErgΔEx4/ΔEx4 knockout mice are embryonic lethal, which is associated with a marked reduction in endocardial-mesenchymal transformation (EnMT) during cardiac valve morphogenesis. We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

Galectin-3: a biologic marker and diagnostic aid for chordoma.

Galectin-3 is a beta-galactoside binding protein. Its expression is quantitatively and qualitatively altered during self-proliferation, malignant transformation, and tumor progression. Galectin-3 is a lectin-related molecule. Lectins are proteins that bind specific carbohydrate structures. Although their precise biologic function is unclear, the general idea is that these molecules operate in modulating cell-to-cell and cell-to-matrix interactions. Galectins have been implicated in cell growth and differentiation and seem to play a role in malignant transformation and metastasis. Galectin-3 is expressed in primitive notochord. The purpose of the current investigation was to identify an immunohistochemical marker to help distinguish the pathologically overlapping entities of chordoma from myxoid low-grade chondrosarcoma. Twelve of 16 (75%) chordomas stained positive for Galectin-3 whereas only one of 12 low-grade myxoid chondrosarcomas stained positive. Galectin-3 chordoma staining is 75% sensitive and 92% specific.

Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol.

Stable nitroxide radicals have been considered as therapeutic antioxidants because they can scavenge more toxic radicals in biologic systems. However, as radicals they also have the potential to increase oxidant stress in cells and tissues. We studied the extent to which this occurs in cultured EA.hy926 endothelial cells exposed to the nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl). Tempol was rapidly reduced by the cells, as manifest by an increase in the ability of the cells to reduce extracellular ferricyanide and by disappearance of the Tempol EPR signal. Cells loaded with ascorbic acid, which directly reacts with Tempol, showed increased rates of Tempol-dependent ferricyanide reduction, and a more rapid loss of the Tempol EPR signal than cells not containing ascorbate. In this process, intracellular ascorbate was oxidized, and was depleted at lower Tempol concentrations than was GSH, another important intracellular low molecular weight antioxidant. Further evidence that Tempol concentrations of 100–1000 μM induced an oxidant stress was that it caused an increase in the oxidation of dihydrofluorescein in cells and inhibited ascorbate transport at concentrations as low as 50–100 μM. The presence of intracellular ascorbate both prevented dihydrofluorescein oxidation and spared GSH from oxidation by Tempol. Such sparing was not observed when GSH was depleted by other mechanisms, indicating that it was likely due to protection against oxidant stress. These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid.

In vivo macrophage recruitment by murine intervertebral disc cells.

SUMMARY An in vivo murine experiment was conducted to measure the capacities of viable intervertebral disc cells to recruit inflammatory cells. The objective was to determine whether compounds secreted from viable cells induce inflammation or whether inflammation in disc herniation simply requires exposure to structural cell or matrix components. Three tissue preparations were inserted into the right lower peritoneal cavity of male mice: tissue with viable annulus fibrosus and nucleus pulposus cells, tissue with viable annulus fibrosus cells, or devitalized annulus fibrosus and nucleus pulposus tissue. Controls included sham-operated and nonoperated groups. Mice were killed 1, 2, or 7 days after surgery. Macrophage recruitment occurred after exposure to viable disc tissue but not after exposure to devitalized disc components; recruitment increased over time. Viable disc cells play a role in the etiology of inflammation in disc herniation.

Growth factor modulation of distraction osteogenesis in a segmental defect model.

A model was established in 39 dogs to investigate the growth factor modulation of regenerate bone in distraction osteogenesis. A segment of the diaphysis of the radius was resected unilaterally. An osteotomy was made proximal to the segmental defect to create a transport segment. A monolateral external fixator was applied. After a latency period, the segment was transported across the defect. One week after the transport assembly contacted the distal pin clamp, an ipsilateral osteotomy of the proximal ulna was performed. In 20 dogs, transforming growth factor-beta was injected into the regenerate bone halfway through the transport period. Four dogs were sacrificed before docking, when the regenerate bone was still immature. In specimens harvested halfway through the transport period, evidence was found of intramembranous ossification during distraction. In specimens harvested after the transport assembly contacted the distal pin clamp, evidence was found that the mature regenerate formed by endochondral ossification. Therefore, a combined mechanism of ossification is proposed for this segmental defect model that includes mechanical stimulus for bone differentiation. The one-time administration of transforming growth factor-beta retarded the formation of a stable, united regenerate. It is concluded that transforming growth factor-beta caused an effect opposite to that which was desired.

Effects of thiol reagents on glucose transport in thymocytes

Rat thymocytes were incubated with 3-O-[14C]methyl D-glucose for 1 h and diluted 100X and the efflux was followed for 1 h. In control cells, about half of the methyl glucose efflux was rapid (t 1/2 approximately 3 min) and about half was slow (t 1/2 congruent to 40 min). The fast and slow compartments represent active and quiescent cells, respectively. A physiological mixture of amino acids present during the loading period dramatically increased the amount of methyl glucose exiting rapidly at the expense of that exiting slowly. Further studies revealed that cysteine was entirely responsible for the action. Cysteine (0.06 mM), glutathione (0.5 mM) and dithiothreitol (0.02 mM) added after completion of fast-phase exit, stimulated subsequent exit about 3-4-fold with no detectable delay. This action was inhibited by catalase and mimicked by 0.04 mM H2O2 and by 0.03 mM N-ethylmaleimide. It did not require extracellular or intracellular Ca2+. These effects are analogous to those seen in adipocytes, implicating sulfhydryl groups in glucose transport regulation [12]. Sulfhydryl oxidation may be a late event in the chain of events leading to glucose transport stimulation by physiological agents.

Purification and Characterization of Canine Collagens I and II

Abstract The purpose of this study was to demonstrate that commercially available human anti-collagen antibodies are suitable for use with canine tissues. This paper describes a reproducible procedure for isolating and purifying canine skin and cartilage collagens in quantities sufficient to characterize. The skin and cartilage were readily dissolved in dilute acetic acid with pepsin, and the collagens were purified by salt precipitation. Purified collagens were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunodetection on electroblot. The tissue distribution of the canine collagens was examined by immunohistochemistry. Two polypeptide chains were identified by SDS-PAGE in the canine skin sample and a human collage Type I control. One polypeptide chain was identified in the canine articular cartilage sample and a human collagen Type II control. Immunoblotting revealed the individual components for the collagen antigens. Anti-human Type II collagen immunoreacted specifically with cells of a chondrocytic phenotype. Antihuman Type I collagen appeared to be less specific. Because the antibodies exhibited cross-species reactivity, homology between canine Type I and II collagens and human Type I and II collagens is suggested. Commercial antibodies for human collagens Type I and II are suitable for study of these canine collagens. (The J Histotechnol 24:51, 2001) Submitted: April 5, 2000; Accepted with revisions: January 5, 2001