Controlled release of bone morphogenetic protein-2 enhances recruitment of osteogenic progenitor cells for de novo generation of bone tissue.

The objective of this study was to evaluate the cellular contribution to the phenomenon of de novo generation of bone tissue induced by the controlled release of bone morphogenetic protein-2 (BMP-2). Gelatin hydrogels (2 mg) incorporating BMP-2 (3 microg) with different water contents were subcutaneously implanted into the back of enhanced green fluorescent protein-chimeric mice to induce the ectopic de novo generation of bone tissue. The hydrogels incorporating BMP-2 could release BMP-2 at different time profiles. When evaluated radiologically and histologically, the ectopic de novo generation of bone tissue was induced by the controlled release of BMP-2 from the hydrogels around the hydrogel-implanted site. The relative percentage number of green fluorescent protein- to osteocalcin-positive cells recruited into the de novo generated bone tissue depended on the BMP-2 release profile. The higher the percentage, the stronger was the de novo generation of bone tissue. These findings indicate that bone marrow-derived osteoblast progenitor cells were recruited from the blood circulation by BMP-2 release and consequently contributed to the ectopic de novo generation of bone tissue. It is conceivable that the local concentration of BMP-2 modifies the recruitment profile of progenitor cells with an osteogenic potential around the release site of BMP-2, resulting in regulated volume of de novo generated bone tissue.

Anti-tumor activity of carmofur water-solubilized by lactic acid oligomer-grafted pullulan nanogels.

Nanogels of pullulan grafted with L-lactic acid oligomer (LLA-o) were prepared to allow a lipophilic anti-tumor drug, carmofur, to solubilize in water. The number of LLA-o grafted was increased with an increase in the amount of LLA-o added in preparation. The LLA-o-grafted pullulan showed critical aggregation concentrations and had 20-100 nm in diameter depending on the grafted number. The carmofur was water-solubilized by the LLA-o-grafted pullulan self-assemble (nanogel), and inhibited the in vitro growth of tumor cells to a similar or high extent compared with carmofur solution in dimethyl sulfoxide (DMSO). When incubated with the spheroid of tumor cells, the nanogel with carmofur showed a strong suppressive effect of tumor cells grown, in contrast to the carmofur solution. When intratumorally injected into mice carrying a tumor mass, carmofur water-solubilized by the nanogel showed stronger suppressive effect of tumor cells growth. It is concluded that the nanogel is a promising carrier to water-solubilize carmofur and enhance the in vitro and in vivo anti-tumor effects.

Prevention of pulmonary metastasis from subcutaneous tumors by binary system-based sustained delivery of catalase.

Catalase delivery can be effective in inhibiting reactive oxygen species (ROS)-mediated acceleration of tumor metastasis. Our previous studies have demonstrated that increasing the plasma half-life of catalase by pegylation (PEG-catalase) significantly increases its potency of inhibiting experimental pulmonary metastasis in mice. In the present study, a biodegradable gelatin hydrogel formulation was used to further increase the circulation time of PEG-catalase. Implantation of (111)In-PEG-catalase/hydrogel into subcutaneous tissues maintained the radioactivity in plasma for more than 14 days. Then, the effect of the PEG-catalase/hydrogel on spontaneous pulmonary metastasis of tumor cells was evaluated in mice with subcutaneous tumor of B16-BL6/Luc cells, a murine melanoma cell line stably expressing luciferase. Measuring luciferase activity in the lung revealed that the PEG-catalase/hydrogel significantly (P<0.05) inhibited the pulmonary metastasis compared with PEG-catalase solution. These findings indicate that sustaining catalase activity in the blood circulation achieved by the use of pegylation and gelatin hydrogel can reduce the incidence of tumor cell metastasis.

Development of a novel antimicrobial peptide, AG-30, with angiogenic properties.

The utility of various synthetic peptides has been investigated in clinical trials of the treatment of cancers, infectious diseases and endocrine diseases. In the process of functional gene screening with in silico analysis for molecules with angiogenic properties, we generated a small peptide, angiogenic peptide (AG)-30, that possesses both antimicrobial and pro-inflammatory activities. AG-30 has an alpha-helix structure with a number of hydrophobic or net positively charged amino acids and a propensity to fold into amphipathic structures. Indeed, AG-30 exhibited antimicrobial activity against various bacteria, induced vascular endothelial cell growth and tube formation in a dose-dependent manner and increased neovascularization in a Matrigel plug assay. As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells. To further evaluate the angiogenic effect of AG-30 in vivo, we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres. In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody). These data suggest that the novel peptide, AG-30, may have therapeutic potential for ischaemic diseases.

Cationized catalase-loaded hydrogel for growth inhibition of peritoneally disseminated tumor cells.

A previous study demonstrated that ethylenediamine-conjugated catalase (ED-catalase) inhibits peritoneal dissemination of tumor cells in mice. To increase its inhibitory effects by sustained release, a hydrogel formulation of ED-catalase was prepared using a biodegradable hydrogel consisting of an acidic gelatin with an isoelectric point of 5.0. Although intraperitoneally injected ED-catalase solution rapidly disappeared from the cavity, more than 10% of ED-catalase remained even at 14 days after implantation of ED-catalase/hydrogel into the cavity. Then, the effect of ED-catalase/hydrogel on peritoneal dissemination of tumor cells was evaluated by measuring the luciferase activity of abdominal organs after intraperitoneal inoculation of colon26/Luc, a colon adenocarcinoma stably expressing luciferase. ED-catalase/hydrogel showed a significantly (P<0.05) greater effect on inhibiting the growth of tumor cells than ED-catalase solution, demonstrating the importance of the retention of ED-catalase within the cavity as far as inhibition is concerned. Serial in vivo images of luciferase activity revealed that the ED-catalase/hydrogel significantly (P<0.05) retarded the growth rate of tumor cells. Survival of tumor-bearing mice supported the findings obtained with the luminescence-based analyses. These findings indicate that the sustained release of ED-catalase from hydrogels into the cavity is highly effective in inhibiting the growth of peritoneally disseminated tumor cells.

Prognostic implication of p53 protein expression in relation to nuclear pleomorphism and the MIB-1 counts in breast cancer.

BACKGROUND: A close correlation of the p53 protein expression to nuclear pleomorphism and proliferative activity in breast cancer has been reported. The prognostic implications of p53 protein expression, however, in relation to nuclear pleomorphism and proliferative activity in breast cancer remain controversial. PATIENTS AND METHODS: Nuclear pleomorphism and immunohistochemical reactivity for p53 protein and MIB-1 were evaluated on formalin-fixed paraffin-stored sections from 250 patients with breast cancer for whom the median follow-up duration was 6.4 years. RESULTS: p53 protein expression was positive in 66 (26.4%) of 250 cases. Nuclear pleomorphism was grade I or II in 169 (67.6%) cases and grade III in 81(32.4%)cases. The MIB-1 counts were more than 10% in 102 (40.8%) cases and less than 10% in 148 (59.2%) cases. There was a close correlation between p53 protein expression and nuclear pleomorphism (p<0.0001) and between p53 protein expression and MIB-1 counts (p<0.0001). Univariate analyses showed the 66 cases with positive p53 protein expression to have a significantly (p=0.0284) worse disease free survival (DFS) than the 184 cases with negative p53 protein expression. A multivariate analysis, however, on the variables including all of p53 protein expression, nuclear pleomorphism and MIB-1 counts indicated the MIB-1 counts (p=0.0041) as well as the lymph node status to be independently significant factors for DFS, while neither p53 protein expression nor nuclear pleomorphism were independently significant factors for DFS. CONCLUSION: The present study demonstrated that the p53 protein expression, nuclear pleomorphism and MIB-1 counts all demonstrated prognostic significance for breast cancer, while the most significant prognostic indicator among these three biological parameters was the MIB-1 counts.