Toru Funayama

Photodynamic Therapy with Indocyanine Green Injection and Near-Infrared Light Irradiation Has Phototoxic Effects and Delays Paralysis in Spinal Metastasis

OBJECTIVE The purpose of this study was to investigate the phototoxic effects of photodynamic therapy (PDT) with indocyanine green (ICG) and near-infrared light irradiation on rat mammary adenocarcinoma cells, and its therapeutic efficacy in a rat model of spinal metastasis. BACKGROUND DATA Although PDT has been successfully used as a non-radiation treatment for many malignancies, it has not yet been clinically applied for treating spinal metastasis. METHODS For the phototoxicity study, CRL-1666 cells were treated with PDT and cell viability was measured by WST-1 assay. For the efficacy study, 26 female Fischer 344 rats with spinal metastasis in the L6 vertebra were divided into three treatment groups: PDT with local injection of ICG (9 rats), PDT with systemic injection of ICG (10 rats), and no treatment or control (7 rats). Both the PDT groups received near-infrared light irradiation with a total energy of 10 J (1 W for 10 sec). The light was delivered directly through a single silica probe which was set on the left side of the L6 vertebral body. Hindlimb motor function was monitored according to the Basso-Beattie-Bresnahan (BBB) scale. Further, the observation periods were calculated to determine the survival time. RESULTS The PDT exerted immediate and persistent phototoxic effects. Furthermore, the PDT with local injection of ICG as well as systemic injection of ICG delayed the deterioration of paralysis and prolonged the observation period. CONCLUSIONS PDT with ICG injection and near-infrared light irradiation could be an effective local adjuvant treatment for spinal metastasis.

Intraoperative Near-infrared Fluorescence Imaging with Novel Indocyanine Green-Loaded Nanocarrier for Spinal Metastasis: A Preliminary Animal Study

Marginal resection during resection of a spinal metastasis is frequently difficult because of the presence of important tissues such as the aorta, vena cava, and dura mater, including the spinal cord adjacent to the vertebral body. Thus, there is an urgent need for novel intraoperative imaging modalities with the ability to clearly identify bone metastasis. We have proposed a novel nanocarrier loaded with indocyanine green (ICG) (ICG-lactosome) with tumor selectivity attributable to its enhanced permeation and retention (EPR) effect. We studied its feasibility in intraoperative near-infrared (NIR) fluorescence diagnosis with ICG-lactosome for imaging spinal metastasis. A rat model of subcutaneous mammary tumor and a rat model of spinal metastasis of breast cancer were used. Fluorescence emitted by the subcutaneous tumors and the spinal metastasis were clearly detected for at least 24 h. Moreover, imaging of the dissected spine revealed clear fluorescence emitted by the metastatic lesion in the L6 vertebra while the normal bone lacked fluorescence. This study was the first report on NIR fluorescence imaging of spinal metastasis in vivo. NIR fluorescence imaging with ICG-lactosome could be an effective intraoperative imaging modality for detecting spinal metastasis.

Bone Regeneration and Remodeling within a Unidirectional Porous Hydroxyapatite Bone Substitute at a Cortical Bone Defect Site: Histological Analysis at One and Two Years after Implantation

Unidirectional porous hydroxyapatite (UDPHAp) is an artificial bone substitute with a unique microstructure consisting of 100–300-µm oval pores that present the material unidirectionally. UDPHAp has a compression strength of 14 MPa and a porosity of 75%, which promotes cell migration and capillary formation within the material. Despite these advantageous properties, bone remodeling and bone formation with UDPHAp remain unclear. To examine long-term remodeling and differences in bone formation based on the defect site, trapezoidal prism-shaped UDPHAp blocks were implanted into rectangular-shaped cortical bone defects in the proximal tibia of Japanese white rabbits. Histological analysis performed at 52 and 104 weeks after implantation revealed that bone and capillaries had formed within the implanted UDPHAp material. Bone formed within the UDPHAp implanted in the cortical defect of rabbit tibia and remodel up to two years. The percentage of new bone area within UDPHAp was larger in cortical lesions than that in medullary lesions. These findings suggest that UDPHAp is a promising material for the repair of non-critical-sized cortical bone defects.

Tanycytic ependymoma of the filum terminale associated with multiple endocrine neoplasia type 1: first reported case

BACKGROUND CONTEXT Ependymoma associated with multiple endocrine neoplasia type 1 (MEN-1) is an extremely rare clinical entity. To the best of our knowledge, only five cases of ependymoma associated with MEN-1 have been previously described. Furthermore, there has been no case of tanycytic ependymoma of the filum terminale associated with MEN-1. PURPOSE The present case report illustrates a 53-year-old man with tanycytic ependymoma of the filum terminale associated with MEN-1. We review the literature on ependymoma with MEN-1 and tanycytic ependymoma of the cauda equina region and also discuss the risk of recurrence. STUDY DESIGN A case report. METHODS The patient presented with complaints of nocturnal pain in the lower back, accompanied by numbness around the anus and intermittent claudication for approximately 1 year. Magnetic resonance imaging (MRI) identified an intradural-enhancing, large mass lesion at the level from Th12 to L2 vertebrae, with a cranial cystic lesion. RESULTS Open-door laminoplasty of the Th12, L1, and L2 and en bloc tumor resection with thickened filum terminale were performed. Histopathologic examination of the tumor specimens showed tanycytic ependymoma (World Health Organization Classification Grade II). At the time of the 2-year and 8-month follow-up examination, MRI did not show tumor recurrence. CONCLUSIONS This is the first reported case of this clinical entity. A careful follow-up of patients with this unusual tumor is strongly recommended.

Multiple capillary hemangiomas of the cauda equina at a level of a single vertebra

Vascular lesions form about 6%–7% of all intradural spinal tumors. The most common vascular tumors of the lumbar spine that involve the conus medullaris and cauda equina regions are pathologically classifi ed as cavernous angioma or cavernoma. Capillary hemangioma of the cauda equina is an extremely rare lesion. Because this tumor has a nonspecifi c appearance on magnetic resonance imaging (MRI) scans, it cannot be accurately differentiated from neurinoma preoperatively. We report a case of intradural capillary hemangiomas of the cauda equina with local multiplicity. The patient was informed that data from the case would be submitted for publication and gave his consent.

Tumor-selective near-infrared photodynamic therapy with novel indocyanine green-loaded nanocarrier delays paralysis in rats with spinal metastasis

BACKGROUND Although recent advances in surgery have improved the quality of life of patients with spinal metastasis, local recurrence is still relatively common. Therefore, there is an urgent need for new surgical treatment options for metastatic spinal cancer. We previously described a novel nanocarrier loaded with indocyanine green (ICG), ICG-lactosome which exhibits tumor selectivity and is a potential near-infrared (NIR) fluorescence imaging agent for the diagnosis of spinal metastasis. The purpose of this study was to investigate the therapeutic effects of tumor-selective photodynamic therapy (PDT) with ICG-lactosome and NIR light irradiation in a rat model of spinal metastasis. METHODS Twenty-one Fischer 344 rats each with a single spinal metastasis in the L6 vertebral body were divided into 3 treatment groups: PDT with a low-concentration ICG-lactosome injection (6 rats), PDT with high-concentration ICG-lactosome injection (7 rats), and a group without ICG-lactosome injection (8 rats). All the animals received local NIR light irradiation with a total energy of 5 J (0.5 W for 10s). RESULTS Both the PDT groups injected with ICG-lactosome showed delayed deterioration of hind-limb paralysis compared with the group without ICG-lactosome. CONCLUSION This modified PDT procedure could be an effective local treatment for spinal metastasis.

A Novel Unidirectional Porous Hydroxyapatite Cylinder Implanted in the Dorsal Muscles of Dogs Promotes Fibrous Tissue Vascularization and Invasion

We recently synthesized a novel unidirectional porous hydroxyapatite (UDPHAp) material with a microstructure consisting of cross-sectional oval pores (diameter, 100-300 μm) . The unidirectional pores of UDPHAp are expected to facilitate the ingrowth of new tissues at sites of implantation. Here, we estimated the osteoinductive capacity of UDPHAp following its implantation in the dorsal muscles of dogs, and also investigated the affinity of UDPHAp for muscle and connective tissues. As a reference material, the HAp porous ceramic product Apaceram® (HOYA, Tokyo, Japan), which is commercially available in Japan and has a different microstructure from UDPHAp, was also used. A cylinder-shaped UDPHAp block was implanted in the dorsal muscles of two beagle dogs. At 1 and 2 years post-implantation, muscle and connective tissues had directly attached to UDPHAp at the upper and lower perforated surfaces. Histological assessment, revealed the direct invasion of fibrous tissues and small capillaries into the unidirectional pores of UDPHAp. Notably, no osseous tissue had formed within UDPHAp. Our findings suggest that the unidirectional pores of UDPHAp are advantageous for the vascularization and invasion of fibrous tissues. However, this unique structure does not contribute to osteoinductive capacity.

Histological Analysis of Bone Bonding and Ingrowth into Connected Porous Hydroxyapatite Spacers in Spinal Surgery

To evaluate the osteoconductive potential of connected porous hydroxyapatite (HAp), we histologically analyzed the newly formed bone inside unidirectional porous HAp (Regenos®, Kuraray, Japan; 75% porosity, n=17) and interconnected porous HAp (Neobone®, Covalent Materials, Japan; 75% porosity, n=10) 26 weeks after their implantation as bone spacers between the split lumbar laminae of goats. As a control, non-connected porous HAp spacers (Apaceram®, Pentax, Japan; 50% porosity, n=5) were used. After staining non-decalcified samples with Villanueva Goldner, changes in pore shape were evaluated microscopically and new bone formation in HAp spacers was quantitatively analyzed. In addition, blood vessel distribution was evaluated by hematoxylin and eosin staining. Changes in pore shape were observed in 76% of the Regenos® spacers and 90% of the Neobone® spacers but were not detected in the Apaceram® spacers. Only limited new bone formation was observed in the Regenos® and Neobone® spacers, whereas vascular-like structures were detected in 82% of the Regenos®, 70% of the Neobone®, and 80% of the Apaceram® spacers. The changes in pore shape were thought to have resulted from the low initial compression strength of the connected porous HAp, which may have limited the inherent osteoconductive potential of connected HAp. Our findings suggest that the maintenance of pore shape is required for promoting new bone formation in connected porous HAp when used as lamina spacers in spinal surgery.

Unidirectional Porous β-Tricalcium Phosphate Bone Substitute: Examination of Balance between New Bone Formation and Absorption

In the present study, we have newly developed an artificial bone substitute, which is unidirectional porous β-tricalcium phosphate (UDPTCP). The objective of this study was to examine the effects of high and low porosity substitutes on the balance between new bone formation and β-TCP absorption. Materials and MethodsSix male Japanese white rabbits (weight 3.1–3.5 kg, approximately 18– 21 weeks old) were used for this study. Intra-venous injection of pent barbiturate was administered and the both medial and lateral femoral condyle were exposed. A hole of 5 mm diameter was drilled to a depth of 12 mm in the metaphysis, perpendicular to the long axis of the femur. (Figure 1) Figure 1. Operation procedureIn the next step, a cylindrical UDPTCP test piece measuring 4.8 × 11 mm was implanted in the holes. Within the bone substitute, unidirectional pores ranging from 100 to 300 μm in diameter were made. This unique architecture fostered transmission of fluids and cells into the piece. In this case, the test piece was implanted into the bone perpendicular to the long axis of the femur, and the orientation of uni-directional pore was parallel to the long axis of femur. We prepared two different test pieces having low (69%) and high (74%) porosities. Half of the animals were sacrificed at 3 weeks after the operation and the remaining half at 6 weeks. After removal of the femoral condyle, the specimen was fixed in formalin and demineralized. Specimens were obtained from the central axis of the cylindrical piece as well as from the lateral or medial surfaces at a distance of 4 mm from midline. The histological samples were prepared for H&E and TRAP staining. Results and Discussion 　At 3 weeks interval, woven bone, which was formed along the wall of the substitute, could be observed by H&E staining in both low and high porosity substitutes (Figure 2a, 2b). In addition, there were osteoblast-like cells lining the newly formed bone surface with extensive capillary formation (Figure 3). At 6 weeks, the β-TCP walls had thinned and bone had matured in both the groups (Figure 4a, 4b). However, in the high-porosity group, β-TCP absorption tended to be more prominent (Figure 4). In addition, it was observed that at the center of the piece, β-TCP absorption was more prominent than that in the 4 mm-area obtained from the lateral or medial surfaces. At 3 and 6 weeks interval, activities of osteoclast-like multinuclear cells were seen on the surface of the pore wall as observed by TRAP staining. Figure 2a. Low porosity (69%) Figure 2b. High porosity (74%) Fig.2a and Fig.2b H&E staining (×12.5) after 3 weeks (center of the specimen)Figure 3. Formation of woven bone with osteoblast-like cells lining the low porosity specimen at 3 weeks. (H&E staining ×400) Figure 4a. Low porosity Figure 4b. High porosityFig.4a and Fig. 4b H&E staining at 6 weeks after implantation. In high porosity, dense-pink staining areas are located at peripheral in the field.Figure 5. TRAP-positive multinuclear cells (black arrow) were seen on the wall and in the capillaries.Conclusions　The UDPTCP implanted in the medullar canal of the femur was absorbed by multinuclear cells and quickly replaced by the newly formed bone. Our results are consistent with those of other studies using porous β-TCP [1]. In our preparation, porosity had certain effects on the balance between bone formation and　β-TCP absorption. Because of the unique architecture of unidirectional pores within the β-TCP specimen as well as easy formation of capillary network and access to osteoclasts may have accelerated absorption of the substitute. UDPTCP is very promising scaffolding material for bone regeneration. However, optimization of the porosity of UDPTCP in accordance with its application site is necessary before its clinical use. Reference[1] Naoki Kondo, Akira Ogose, Kunihiko Tokunaga, Tomoyuki Ito, Katsumitsu Arai, Naoko Kudo, Hikaru Inoue, Hiroyuki Irie, Naoto Endo: Bone formation and resorption of highly purified β-tricalcium phosphate in the rat femoral condyle. Biomaterials 26: 5600-5608, October 2005.

Study of the Mechanical Properties of a Novel Unidirectional Porous Hydroxyapatite Implanted in the Femoral Marrow of a Rabbit

The objective of this study was to assess the long-term stability and mechanical strength of a novel unidirectional porous hydroxyapatite (UDPHAp). Thirty-five Japanese white rabbits were used in the study. A cylindrical piece of UDPHAp was implanted in the femoral marrow. The animals were sacrificed 6, 12, 52 and 104 weeks after implantation. Compression strength was measured in a direction parallel to that of the unidirectional pores. The mean compression strength of the control sample before implantation was 13.4 MPa. The mean compression strength was 29.5 MPa, 43.5 MPa, 49.2 Mpa, 39.4 MPa and 45.8 MPa at 6, 12, 26, 52 and 104 weeks after implantation, respectively. Statistically significant differences were observed between the control samples and the implanted samples at all time points and between the implanted samples tested at 6 and 26 weeks after implantation. UDPHAp offers an advantage with regard to mechanical strength in that the compression strength in the direction parallel to that of the unidirectional pores increases at an early stage after implantation in the living bone tissue and is maintained for at least 2 years.