Because bone morphogenetic protein 2 gene transfected Escherichia coli (E-BMP-2) produce recombinant human BMP-2 (rhBMP-2) more efficiently than mammalian cells (Chinese hamster ovary [CHO]-BMP-2), they may be a more cost-effective source of rhBMP-2 for clinical use. However, use of E-BMP-2 for regenerating long bones in large animals has not been reported. In the current study, we evaluated the healing efficacy of E-BMP-2 in a canine model. We created 2.5-cm critical-size segmental ulnar defects in test animals, then implanted E-BMP-2 and 700 mg of artificial bone (beta-tricalcium phosphate; β-TCP) into the wounds. We examined the differential effects of 5 E-BMP-2 treatments (0, 35, 140, 560, and 2240 μg) across 5 experimental groups (control, BMP35, BMP140, BMP560, and BMP2240). Radiography and computed tomography were used to observe the regeneration process. The groups in which higher doses of E-BMP-2 were administered (BMP560 and BMP2240) displayed more pronounced bone regeneration; the regenerated tissues connected to the host bone, and the cross-sectional areas of the regenerated bone were larger than those of the originals. The groups in which lower doses of E-BMP-2 were administered (BMP35 and BMP140) experienced relatively less bone regeneration; furthermore, the regenerated tissues failed to connect to the host bone. In these groups, the cross-sectional areas of the regenerated bone were equal to or smaller than those of the originals. No regeneration was observed in the control group. These findings suggest that, like CHO-BMP-2, E-BMP-2 can be used for the regeneration of large defects in long bones and that its clinical use might decrease the cost of bone regeneration treatments.

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When the usage of hydroxyapatite (HAp) was first approved at clinics by the Kouseishou (Japanese FDA) as a bone substitute (APACERAM), the upper limit of pore content was set at 60%. Cells play an important role in bone repair, especially in regeneration therapy, but on using these HAps, the cells cannot penetrate deeply into them because their inside pores rarely connect. To promote cell penetration into the inside of the HAps, we have developed superporous HAps (HAp-Ss). First, phosphoric acid was added to a calcium hydroxide solution, and the mixture was dried by the spray-dry method to produce fine primary particles. Then, two kinds of surfactants were used to form a large amount of pores. These two HAp-Ss have 85% porosity and interconnected pores in the inside. They were tested with a culture of primary rat osteoblasts, which showed good penetration therein. The penetrated osteoblasts maintained high alkaline phosphatase activity during the culture period. This indicates that the developed HAp-Ss are very good bone substitutes and also useful scaffolds in bone regeneration therapy.

Morphological studies of secondary palate formation, with special reference to the development of rugae, were carried out on Jcl:ICR mouse embryos. Three rugae were observed on the anterior part of the future oral surface of the vertically developing palatal shelves in 13-day embryos. Rugae increased in number as the development of the palatal shelves proceeded, and five to six prominent rugae were observed in 14-day embryos just prior to shelf elevation. The folding of these five to six rugae progressed in conjunction with the formation of a sharp, valley-like groove at the base of the anterior two-fifths of the vertical palatal shelves. As palatal shelves elevated, the groove disappeared gradually, and, accordingly, the folding of rugae loosened. In the groove region, the superficial epithelial cells were roundish, while the basal ones were elongated. Such characteristic features were no longer observed when the disappearance of the groove was completed. Eight rugae were observed on the future hard palate of 14-day embryos with already completed palatal fusion. An additional ruga was frequently found in 15-day embryos, and the pattern then was almost the same as that of an adult. Epithelial thickening and condensation at the rugae region, as well as mesenchymal condensation under the epithelium of the rugae, were confirmed in embryos both before and after elevation of the palatal shelves. There is a possibility that these structural characteristics observed in the epithelial and mesenchymal cells of the rugae and groove regions may be related to palatal shelf elevation.

Telomeres protect chromosome ends from being recognized as DNA double-strand breaks. Telomere shortening, which occurs due to incomplete replication of DNA termini, limits the proliferative capacity of human somatic cells and contributes as a barrier to carcinogenesis. In most human cancer cells, telomerase maintains telomere length whereas TRF1, a telomeric protein, represses telomere access to telomerase. Tankyrase 1 is a PARP that dissociates TRF1 from telomeres by poly(ADP-ribosyl)ating TRF1. Thus, by reducing TRF1 loading on chromosome ends, tankyrase 1 enhances telomere access to telomerase and causes telomere elongation. Recent studies of knockout mice suggest that tankyrases may not regulate telomere length in mice (Mus musculus). Consistent with this idea is that mouse TRF1 has no canonical tankyrase-binding motif. However, the presence of such a motif is not a prerequisite to bind tankyrase 1 in certain species. Here, we found that, in mice, tankyrase 1 does not bind or poly(ADP-ribosyl)ate TRF1. Accordingly, mouse TRF1 was resistant to tankyrase 1-mediated release from telomeres. These observations indicate that telomeric function of tankyrase 1 is not conserved in mice. We also found that the canonical tankyrase 1-binding motif in TRF1 is conserved in several mammals but not in rats. Since mice and rats have much higher telomerase activity in their somatic tissues and much longer telomeres than those in other mammals, these rodent species might have evolved to resign the tankyrase 1-mediated telomere maintenance system. Meanwhile, PARP inhibitors induced non-telomeric tankyrase 1 foci in the nuclei, suggesting another function of tankyrase 1 at non-telomeric loci. (Cancer Sci 2007; 98: 850-857)