Atsumasa Uchida

Calcium hydroxyapatite ceramic used as a delivery system for antibiotics

Porous blocks of calcium hydroxyapatite ceramic were evaluated as delivery systems for the sustained release of antibiotics. We tested gentamicin sulphate, cefoperazone sodium, and flomoxef sodium in powder form placed in a cylindrical cavity in calcium hydroxyapatite blocks, using in vitro studies of elution and in vivo studies in rats. Gentamicin sulphate gave a maximum concentration within the first week, which gradually decreased but was still effective at 12 weeks, when 70% of the antibiotic had been released. Even at this stage the antibiotic concentration from a 75 mg dose was five times the minimum inhibitory concentration for staphylococci. In the in vivo studies the release of gentamicin sulphate into the normal bone of rats was at similar rates and levels. The bacteriocidal activity of the drugs was not affected by packing into calcium hydroxyapatite ceramic and the blocks were completely biocompatible on histology. This new system overcomes the disadvantages of other drug delivery systems, avoiding thermal damage to the antibiotics and a second operation for the removal of the carrier. Some mechanical strength is provided by the ceramic and healing may be accelerated by bone ingrowth into its micropores.

Prognostic significance of Ki‐67 reactivity in soft tissue sarcomas

Proliferative activity of soft tissue sarcomas (STS) in 34 cases was estimated by immunohistochemical procedures (avidin‐biotin complex [ABC] method) with monoclonal antibody Ki‐67 which reacts with a nuclear antigen expressed in all phases of cell cycle except G0. In 20 of 34 cases (59%), varying numbers of Ki‐67‐positive tumor cells were detected with a range from 5 to 382 per 10 high power fields (HPF) (mean 57.2/10 HPF). Ki‐67 index (the number of Ki‐67‐positive tumor cells/10 HPF) positively correlated with mitotic count (r = 0.428, P < 0.02), cellularity (r = 0.447, P < 0.01), and histologic grade (r = 0.473, P < 0.01). The Ki‐67 low index group (less than 50/10 HPF) showed more favorable prognosis than the high index group (more than 50/10 HPF) (P < 0.005). Three cases with low mitotic count and unfavorable prognosis were proved to be the Ki‐67 high index group (142‐382/10 HPF). These results indicated that reactivity of tumor cells for Ki‐67 is a useful prognostic marker in the patients with STS, and might be used as one of the histologic factors for the grading of STS.

Prognostic factors in angiosarcoma : A multivariate analysis of 55 cases

Data for prognostic factors in angiosarcoma (AS) are limited, prompting a large‐scale study of AS with multivariate analysis. To analyze prognostic factors in angiosarcoma (AS), clinical and histologic findings in 55 patients collected from hospitals in Japan were reviewed. Prognostic factors were evaluated by univariate and multivariate Coxs proportional hazards models. The study involved 32 males and 23 females, ages 18–93 (median, 69) years. The primary sites of tumors included head and neck (32 cases), trunk (10), extremities (3), spleen (3), breast (3), and other (4). The overall 2‐year survival rate was 21%. Univariate analysis of clinical factors including age, sex, size and depth of tumor, tumor‐related symptoms, interval between onset of symptoms and admission, surgical procedures, adjuvant chemotherapy, and adjuvant radiotherapy showed that age, tumor size, and mode of treatment were significant for survival. Histologic factors analyzed were mitotic counts, cellularity, cellular pleomorphism, extent of necrosis, vascular differentiation, and nonspecific diagnosis. Only mitotic counts were significant for prognosis. Multivariate analysis on these four factors revealed that tumor size, mode of treatment, and mitotic counts were independent prognostic factors.

The use of calcium hydroxyapatite ceramic in bone tumour surgery

We report 60 benign bone tumours treated by resection and curettage followed by the implantation of calcium hydroxyapatite ceramic (CHA). After follow-up of six to 60 months (average 36), no patient had local recurrence of the tumour or any adverse effects from the implants. In almost all cases radiography showed that the CHA was well-incorporated into the host bone, with new bone formation in and around the CHA. Corrective remodelling of deformed bone and normal fracture healing suggested that there was normal bone turnover in the presence of the CHA. Histology of biopsies from seven patients showed bone ingrowth into the pore structure of CHA in the central zone of some defects by one year after implantation. CHA appears to be a useful substitute for bone graft in the treatment of some benign tumours.

Experimental implant-related osteomyelitis treated by antibiotic-calcium hydroxyapatite ceramic composites

The efficacy of locally implanted antibiotic-calcium hydroxyapatite ceramic composites was investigated for the treatment of experimentally produced, implant-related osteomyelitis in rats. High concentrations of antibiotics were detected at the site of infection and bacteria were eradicated without removal of the metal implants. Parenteral antibiotics and surgical debridement, alone or in combination with antibiotic-impregnated acrylic bone cement, all failed to eradicate the infections.

New ferromagnetic bone cement for local hyperthermia.

We have developed a ferromagnetic bone cement as a thermoseed to generate heat by hysteresis loss under an alternate magnetic field. This material resembles bioactive bone cement in composition, with a portion of the bioactive glass ceramic component replaced by magnetite (Fe3O4) powder. The temperature of this thermoseed rises in proportion to the weight ratio of magnetite powder, the volume of the thermoseed, and the intensity of the magnetic field. The heat-generating ability of this thermoseed implanted into rabbit and human cadaver tibiae was investigated by applying a magnetic field with a maximum of 300 Oe and 100 kHz. In this system, it is very easy to increase the temperature of the thermoseed in bone beyond 50 degrees C by adjusting the above-mentioned control factors. When the temperature of the thermoseed in rabbit tibiae was maintained at 50 to 60 degrees C, the temperature at the interface between the bone and muscle (cortical surface) surrounding the material rose to 43 to 45 degrees C; but at a 10-mm distance from the thermoseed in the medullary canal, the temperature did not exceed 40 degrees C. These results demonstrate that ferromagnetic bone cement may be applicable for the hyperthermic treatment of bone tumors.

Immunohistochemical Detection of bcl-2 and p53 Proteins and Apoptosis in Soft Tissue Sarcoma: Their Correlations with Prognosis

Information on prognostic factors is essential to establish appropriate therapeutic modalities for soft tissue sarcoma (STS). To evaluate the biological nature and prognostic factors of STS, p53 and bcl-2 expression was immunohistochemically studied on paraffin-embedded sections from 70 patients with STS in the extremities and trunk. In addition, the degree of apoptosis was examined by in situ end-labeling. Histologic diagnoses in these cases were malignant fibrous histiocytoma in 29 cases, liposarcoma in 11, synovial sarcoma in 11, leiomyosarcoma in 5, malignant neurogenic tumor in 5, and others in 9. Tumor cells in 31 of 70 cases (44%) showed positive nuclear staining for p53 protein. There was no correlation between p53 expression and tumor size, histologic grade, argyrophilic nucleolar organizer region (AgNOR) count, cellularity and extent of neerosis. Expression of p53 did not correlate with survival of patients. Tumor cells in 24 of 56 cases (43%) were positive for bcl-2 protein expression. The frequency of bcl-2 expression in the tumor cells showed a direct proportion to tumor size (> or = 10 vs. < 10 cm) but inverse proportion to AgNOR counts and cellularity. The 5-year survival rate in patients with bcl-2-positive tumors (87%) was more favorable than in those with bcl-2-negative tumors (53%; p < 0.05). The frequency of apoptosis in low-grade STS was significantly higher than that in the intermediate and high-grade STS (p < 0.001). Extent of necrosis, a well-known prognostic indicator in STS, was not correlated with the frequency of apoptosis. Multivariate analysis showed that cellularity, bcl-2 and AgNOR counts were independent prognostic factors in patients with STS. The current study revealed that STS with a higher expression of bcl-2 had lower proliferative activity and larger size than those without. Immunohistochemical detection of bcl-2 is useful for predicting prognosis in patients with STS.

INFLUENCE OF LOCAL RECURRENCE ON THE PROGNOSIS OF SOFT-TISSUE SARCOMAS

We have investigated the significance of local recurrence on survival in 173 patients with localised soft-tissue sarcomas of the limbs and of the trunk. The overall survival rates at five and ten years were 75.2% and 68.0%, respectively. After definitive surgery at our hospitals, there was local recurrence in 25 patients (14.5%). After inadequate operations elsewhere, there was a higher incidence of late local recurrence (28.3%), in comparison with those with primary tumours treated by us (9.0%), or patients referred to us immediately after inadequate surgery elsewhere (10.2%). Because of small numbers these differences in the survival rates were not statistically significantly different. Univariate survival analysis showed that local recurrence after definitive surgery (p = 0.006) together with the histological grade (p = 0.0002), the size of the tumour (p = 0.002), its depth in relation to deep fascia (p = 0.003), and the surgical margin (p = 0.0001) were the significant prognostic factors. Local recurrence at the initial presentation did not affect survival. Multivariate analysis showed that local recurrence after definitive surgery also lost its apparent prognostic significance.

Smooth Muscle Cell Phenotype-dependent Transcriptional Regulation of the α1 Integrin Gene

The expressional regulation of chicken α1 integrin in smooth muscle cells was studied. The α1 integrin mRNA was expressed developmentally and was distributed dominantly in vascular and visceral smooth muscles in chick embryos. In a primary culture of smooth muscle cells, α1 integrin expression was dramatically down-regulated during serum-induced dedifferentiation. Promoter analyses revealed that the 5′-upstream region (−516 to +281) was sufficient for transcriptional activation in differentiated smooth muscle cells but not in dedifferentiated smooth muscle cells or chick embryo fibroblasts. Like other α integrin promoters, the promoter region of the α1 integrin gene lacks TATA and CCAAT boxes and contains binding sites for AP1 and AP2. The essential difference from other α integrin promoters is the presence of a CArG box-like motif. Deletion and site-directed mutation analyses revealed that the CArG box-like motif was an essential cis-element for transcriptional activation in differentiated smooth muscle cells, whereas the binding sites for AP1 and AP2 were not. Using specific antibodies, a nuclear protein factor specifically bound to the CArG box-like motif was identified as serum response factor. These results indicate that α1 integrin expression in smooth muscle cells is regulated transcriptionally in a phenotype-dependent manner and that serum response factor binding plays a crucial role in this regulation.

Prognostic significance of Ki67 (MIB1) proliferation index and p53 over‐expression in chondrosarcomas

To investigate the prognostic significance of the Ki67 (MIB1)‐proliferation index and p53 over‐expression in chondrosarcomas, we retrospectively analyzed a cohort of 29 patients with chondrosarcomas using immunohistochemical assays with MIB1 and p53 monoclonal antibodies on formalin‐fixed, paraffin‐embedded tissue samples with microwave preparation. We also assessed 19 patients with benign cartilaginous tumors as a control group. There was a significant positive correlation between MIB1 index and tumor grade in chondrosarcomas, while there was no significant difference in the MIB1 index between the grade‐I chondrosarcomas and the benign cartilaginous tumors. Patients categorized in the high‐MIB1‐index group had a significantly lower survival rate than those in the low‐index group. Moreover, in analyzing the sub‐set of the patients with grade‐II chondrosarcomas, it was found that they could be prognostically sub‐divided according to MIB1 index. The p53 index also significantly correlated with patient survival, and there was significant correlation between the MIB1 index and the p53 index. However, in multivariate analysis, only the MIB1 index and tumor grade proved to be independent prognostic indicators of chondrosarcomas. These results demonstrate that the MIB1 index can be a useful procedure for assessing tumor grade in chondrosarcomas, especially for determining the prognosis of patients with grade‐II chondrosarcoma.