R. Nishimura

Tailor-made tricalcium phosphate bone implant directly fabricated by a three-dimensional ink-jet printer

Rapid prototyping (RP) is a molding technique that builds a three-dimensional (3D) model from computer-aided design (CAD) data. We fabricated new tailor-made bone implants (TIs) from α-tricalcium phosphate powder using an RP ink-jet printer based on computed tomography (CT) data, and evaluated their safety and efficacy. CT data of the skulls of seven beagle dogs were obtained and converted to CAD data, and bone defects were virtually made in the skull bilaterally. TIs were designed to fit the defects and were fabricated using the 3D ink-jet printer with six horizontal cylindrical holes running through the implants, designed for possible facilitation of vascular invasion and bone regeneration. As a control, hydroxyapatite implants (HIs) were cut manually from porous hydroxyapatite blocks. Then, craniectomy was performed to create real skull defects, and TIs and HIs were implanted. After implantation, CT was performed regularly, and the animals were euthanized at 24 weeks. No major side effects were observed. CT analysis showed narrowing of the cylindrical holes; bony bridging between the implants and the temporal bone was observed only for TIs. Histological analysis revealed substantial new bone formation inside the cylindrical holes in the TIs, while mainly connective tissues invaded the porous structures in HIs. Bone marrow was observed only in TIs. Osteoclasts were seen to resorb regenerated bone from inside the cylindrical holes and to invade and probably resorb the TIs. These data suggest that TIs are a safe and effective bone substitute, possessing osteoconductivity comparable with that of HIs.

Establishment of four pairs of canine mammary tumour cell lines derived from primary and metastatic origin and their E-cadherin expression.

Four new pairs of canine mammary carcinoma cell lines derived from both primary and metastatic lesions were established. The cells were cultured in RPMI-1640 with 10% fetal bovine serum and they showed stable growth for more than 120 passages. Using these cell lines, the expression of E-cadherin was measured by flow cytometry and the function of E-cadherin was evaluated by cell aggregation assay and results from the primary and metastatic lesions were compared statistically. E-cadherin was strongly expressed in all of the cell lines, without a notable difference between cells of primary and metastatic origin. In the cell aggregation assay, the function of E-cadherin was significantly weaker in the cells of primary origin (p < 0.05), as compared with cells of metastatic origin. The present results suggest that a reduction in E-cadherin function may be implicated in the invasive and metastatic potential of canine mammary tumour cells; however, further study will be needed to clarify E-cadherin function in the context of the metastasis of canine mammary carcinoma.

Bone regeneration within a tailor-made tricalcium phosphate bone implant with both horizontal and vertical cylindrical holes transplanted into the skull of dogs

A new tailor-made bone implant (TI) with six horizontal cylindrical holes fabricated from α-tricalcium phosphate powder, as described in our previous report, was modified to include five additional vertical holes (TI-v) in an attempt to accelerate the bone regeneration through the holes. This TI-v implant and hydroxyapatite implants (HI) as controls were transplanted into experimental skull defects in dogs. Computed tomography (CT) was performed immediately after the surgery and then every 4 weeks. The dogs were killed for histological analysis at 24 weeks of implantation. On CT, bone bridging between the implant and the skull was observed in the TI-v group from 8 weeks of implantation, whereas a clear bone bridge was not formed in the HI group after 24 weeks of implantation. Histological analysis revealed collagen tissues and new bone formation in the horizontal cylindrical holes in most of the TI-v group, whereas mainly connective tissues invaded the porous structures in the HI group. In the Ti-v group, at the middle of the horizontal holes where they crossed the vertical holes, fibrous collagen tissues and muscular tissue filled up the hole and new bone formation seemed to be blocked. However, in the TI-v group more collagen and bone tissues were formed than in the HI group; when compared with the data in our previous report, however, the total volume of regenerated bone in the horizontal cylindrical holes in the TI-v seemed to be less than that in the TI. Thus, the addition of vertical cylindrical holes in the TI-v was not effective in promoting the faster stabilization of the TI-v in the skull of the dog.

Effects of transplantation sites on tumour growth, pulmonary metastasis and ezrin expression of canine osteosarcoma cell lines in nude mice

To determine the influence of the transplantation site of canine osteosarcoma (OS) cell lines on tumour growth and pulmonary metastasis, three OS cell lines were transplanted into nude mice via subcutaneous (SC), intratibial (IT) or intravenous (IV) injection. IT-xenografts exhibited greater potential for developing primary masses and pulmonary metastasis than SC-xenografts. In IT and IV xenografts, lung micrometastases along with phosphorylated ezrin-radixin-moesin (p-ERM) overexpression were found in mice xenografted with HMPOS and OOS cells after 1 week and metastasis was found with decreased p-ERM expression at later time points. The expression of ezrin and p-ERM in the primary tumours of IT-xenografted mice was higher than those in SC-xenografted mice with HMPOS and OOS cells. The results suggest that the orthotopic transplantation site plays an important role in the spontaneous metastasis of canine OS and that ezrin phosphorylation may be involved in the early metastatic mechanism of canine OS cells.

Effects of low-dose cyclophosphamide with piroxicam on tumour neovascularization in a canine oral malignant melanoma-xenografted mouse model

Low-dose cyclophosphamide (CyLD) has shown promise in the treatment of several cancers; however, the effect of CyLD on canine oral malignant melanoma has never been explored. In this study, we investigated the effects of CyLD with or without piroxicam (Px) on tumour neovascularization and vascular normalization in a canine oral malignant melanoma-xenografted mice model. After treatment with CyLD, Px or a combination of both (CyPx), the growth of the tumour in the treatment groups was significantly suppressed compared to the control group at 30 days of treatment. Proliferation index was also significantly reduced by all treatments, only CyPx significantly lowered microvessel density and vascular endothelial growth factor (VEGF) levels. Additionally, CyLD significantly reduced the proportion of normal vessels and caused an imbalance between VEGF and thrombospondin-1. These results suggested that CyPx has potent anti-angiogenic effects in terms of both the number and quality of blood vessels in xenografted canine oral malignant melanoma.

Establishment of a pair of novel cloned tumour cell lines with or without metastatic potential from canine mammary adenocarcinoma.

We produced 23 cloned cell lines from parental CHMp, which was previously established from a canine mammary adenocarcinoma patient in our laboratory. Two representative cloned cell lines, namely, CHMp-5b and -13a, were selected and characterized for cellular morphology, growth potential and expression of some tumour-related proteins. Subsequently, we transplanted the 2 tumour cell lines orthotopically into female nude mice to examine their tumorigenicity and metastatic potential. Interestingly, despite sharing the same origin, only CHMp-5b cells metastasized to the lung. Our results indicate that a comparison between these 2 cell lines at the molecular level will help us understand mechanisms of tumour progression, especially in the context of distant metastases originating from canine mammary gland tumours.

Implantation of tetrapod-shaped granular artificial bones or β-tricalcium phosphate granules in a canine large bone-defect model.

ABSTRACT We investigated biodegradability and new bone formation after implantation of tetrapod-shaped granular artificial bone (Tetrabone®) or β-tricalcium phosphate granules (β-TCP) in experimental critical-size defects in dogs, which were created through medial and lateral femoral condyles. The defect was packed with Tetrabone® (Tetrabone group) or β-TCP (β-TCP group) or received no implant (control group). Computed tomography (CT) was performed at 0, 4 and 8 weeks after implantation. Micro-CT and histological analysis were conducted to measure the non-osseous tissue rate and the area and distribution of new bone tissue in the defect at 8 weeks after implantation. On CT, β-TCP was gradually resorbed, while Tetrabone® showed minimal resorption at 8 weeks after implantation. On micro-CT, non-osseous tissue rate of the control group was significantly higher compared with the β-TCP and Tetrabone groups (P<0.01), and that of the β-TCP group was significantly higher compared with the Tetrabone group (P<0.05). On histology, area of new bone tissue of the β-TCP group was significantly greater than those of the Tetrabone and control groups (P<0.05), and new bone distribution of the Tetrabone group was significantly greater than those of the β-TCP and control groups (P<0.05). These results indicate differences in biodegradability and connectivity of intergranule pore structure between study samples. In conclusion, Tetrabone® may be superior for the repair of large bone defects in dogs.

Effects of etoposide alone and in combination with piroxicam on canine osteosarcoma cell lines

Osteosarcoma (OSA) is the most common primary bone tumour in dogs. The poor survival rate in dogs with OSA highlights the need for new therapeutic approaches. This study evaluated the cytotoxic effects of etoposide, alone and in combination with piroxicam, on canine OSA cell cultures. Etoposide alone significantly suppressed cell growth and viability, whereas etoposide in combination with piroxicam exhibited concentration dependent cytotoxicity. The anti-proliferative effect was a result of inactivity of the Cdc2-cyclin B1 complex, which correlated with an increase in the G2/M fraction. This subsequently activated the apoptosis cascade, as indicated by elevated apoptosis levels and up-regulation of poly (ADP-ribose) polymerase proteolytic cleavage. Down-regulation of survivin expression induced by the combination treatment may have contributed to the enhanced cytotoxicity. The results of this study suggest that further investigation of etoposide and piroxicam as a therapeutic combination for canine OSA is warranted.

Molecular investigation of the direct anti-tumour effects of nonsteroidal anti-inflammatory drugs in a panel of canine cancer cell lines

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been suggested as effective adjunctive anti-tumour agents in human and veterinary medicine. However, the molecular mechanisms associated with their anti-tumour effects and correlations with the expression of cyclooxygenase (COX) and related molecules in tumours remain controversial. The objective of this study was to compare the expression profiles of COX and related molecules with NSAID sensitivity and to explore the molecular mechanisms of anti-tumour effects. The expression profiles of COXs, prostaglandins (PGs), PGD2 synthases, and PGE2 synthases were obtained, and their correlations with in vitro sensitivity to the NSAIDs piroxicam, carprofen, and robenacoxib were examined, using 26 canine cancer cell lines. Subsequently, microarray analysis was performed using one melanoma cell line to gain insight into mechanisms by which NSAIDs could exert cytotoxic effects. No strong correlation was observed between the cellular expression of COX and related molecules and sensitivity to NSAID treatment. Additionally, NSAIDs inhibited cell growth only at considerably higher concentrations than those required for functional COX inhibition. Microarray data demonstrated that five genes (SLC16A6, PER2, SLC9A8, HTR2B, and BRAF) were significantly upregulated and that four genes (LOC488305, H2AFJ, LOC476445, and ANKRD43) were significantly downregulated by NSAID exposure to the melanoma cell line. These results suggest that the direct in vitro anti-tumour effects of NSAIDs might be mediated by COX/PG-independent pathways. Novel candidate genes that could potentially be involved in the anti-tumour effects of NSAIDs were identified. Further validation and elucidation of their associated mechanisms will contribute to patient selection in clinical settings and the development of effective combination therapies.

Anti-tumour efficacy of etoposide alone and in combination with piroxicam against canine osteosarcoma in a xenograft model

Osteosarcoma (OSA) in dogs is locally invasive and highly malignant. Distant metastasis is the most common cause of death. To date, the survival rate in dogs with OSA remains poor. The cytotoxic effects of etoposide against canine OSA cell lines, either alone or in combination with piroxicam, have been previously demonstrated in vitro. The aim of this study was to evaluate the anti-tumour effect of etoposide alone and in combination with piroxicam on canine OSA using murine models. Etoposide single agent treatment significantly delayed tumour progression with a marked reduction in Ki-67 immunoreactivity in tumour tissue. Concomitant treatment with piroxicam did not enhance the anti-tumour efficacy of etoposide. Etoposide single agent treatment and combination treatment with piroxicam down-regulated survivin expression, but was not followed by increased apoptotic activity. These findings indicate that etoposide might be a promising novel therapeutic for canine OSA. Further investigations into its potential for clinical application in veterinary oncology are warranted.