In vivo osteogenic capability of cultured allogeneic bone in porous hydroxyapatite: immunosuppressive and osteogenic potential of FK506 in vivo.

Fischer or ACI rat marrow cells were obtained from femoral shafts and were cultured to confluence in Eagle's minimal essential medium (EMEM) supplemented with 15% fetal bovine serum. After trypsinization, the cells were subcultured on porous hydroxyapatite (HA; Interpore 500) blocks in the presence of beta-glycerophosphate and 10 nM dexamethasone (Dex). After 2 weeks of subculture, a mineralized bone matrix with osteogenic cells developed on the HA pore surfaces. ACI or Fischer cultured bone tissue/HA constructs were implanted subcutaneously into the backs of Fischer rats and the immunosuppressant FK506 was given to the rats for 4 weeks. Implants were harvested 4 weeks and 8 weeks after insertion. At 4 weeks, the ACI constructs (allografts) showed high levels of osteogenic parameters (alkaline phosphatase [ALP] activity and osteocalcin content) and bone formation was observed together with active osteoblasts without obvious accumulation of inflammatory cells. At 8 weeks, active osteoblasts and progressive bone formation were still observed, while osteogenic parameters remained high and osteocalcin messenger RNA (mRNA) was detected. Without FK506 administration, the allografts showed neither bone formation nor osteocalcin mRNA and there were only trace levels of the osteogenic parameters. In the case of Fischer constructs (isografts), extensive bone formation was detected and all the osteogenic parameters were higher with FK506 than without FK506 at both 4 weeks and 8 weeks. These results indicate that cultured bone tissue/HA constructs possess a high osteogenic potential, even as allografts, and that FK506 not only has an immunosuppressive action, but also promotes bone formation.

In vivo osteogenic durability of cultured bone in porous ceramics: a novel method for autogenous bone graft substitution.

BACKGROUND: Bone marrow cells differentiate into bone-forming osteoblasts when cultured in medium supplemented with 15% fetal bovine serum, ascorbic acid, beta-glycerophosphate, and dexamethasone. METHODS: To investigate in vivo osteoblastic activity and bone matrix formation by cultured bone marrow cells, Fischer rat marrow cells were cultured for 2 weeks in porous hydroxyapatite (HA) and then subcutaneously implanted into 7-week-old male syngeneic rats. The implants were harvested after 8 and 52 weeks for biochemical and histological analyses. RESULTS: At both times, formation of lamellar bone accompanied by regeneration of marrow were seen in many of the HA pores. When a fluorochrome (calcein) was administered at 50 weeks after implantation, it was detected in the pores of implants harvested at 52 weeks. Osteoclastic resorption followed by new bone formation was seen in some pores at 52 weeks, indicating that bone remodeling was continuing. The alkaline phosphatase activity of implants harvested at 52 weeks was comparable to that at 8 weeks, whereas the osteocalcin content of the implants harvested at 52 weeks was about twice that at 8 weeks. CONCLUSION: These results demonstrated that there was persistent in vivo osteogenic and hematopoietic activity in the prefabricated bone/HA constructs, and indicated that normal bone tissue was regenerated after grafting of the constructs, which were brittle before implantation. Tissue engineering using HA and cultured marrow cells culture may provide an alternative method of bone transplantation for patients with skeletal disorders, although further in vivo and in vitro experiments are needed.

Carcinoma of the pancreas with endocrine component in childhood. A case report.

A case of pancreatic tumor in a six-year-old girl is presented. The tumor had histologic characteristics of acinar cell carcinoma with endocrine component. Grossly, it was encapsulated and attached to the tail of the pancreas, measuring 8 cm in the greatest diameter. Histologically, the tumor was composed of medium-sized tumor cells, with mild pleomorphism showing mainly acinar structures. Many of these tumor cell contained fine granules that were periodic acid-Schiff positive, diastase resistant, and positive with dimethylaminobenzaldehyde nitrite strain for tryptophan, and some contained granules that were positive with Grimelius stain and positive with peroxidase-antiperoxidase technic for gastrin. Electron microscopy revealed two types of membrane-bound granules in the tumor cells. The larger granules measured 400-700 nm in diameter and appeared to be zymogen granules, while the smaller ones measured 100-200 nm in diameter and appeared to be neuroendocrine granules. Some cells contained both granules. The postoperative course of the patient was excellent, and she was alive and well 13 years after operation. This may be the second reported case of acinar-endocrine cell tumor of the pancreas.

Paraganglioma of the posterior mediastinum: value of magnetic resonance imaging.

A case of paraganglioma arising in the posterior mediastinum in a 29-year-old man diagnosed by magnetic resonance imaging is reported. Excision of mediastinal paraganglioma is often hazardous because of its rich vascular supply and tendency to involve surrounding structures. Magnetic resonance imaging is valuable for the preoperative diagnosis of this vascular tumor as well as for determination of its resectability and appropriate surgical procedure.

Increased expression of extracellular signal regulated kinase 1 after axotomy in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus.

The extracellular signal regulated kinases (Erks) cascade is a major signalling system by which cells transduce extracellular signals into intracellular responses. To obtain information about the role of Erks in retrograde neuronal reaction, we investigated the changes of Erk 1 and Erk 2 with in situ hybridization and immunohistochemical study in the dorsal motor nucleus of vagus nerve, which shows degenerative changes, and the hypoglossal nucleus, which shows regenerative changes, of adult rats after axotomy. The expression of mRNA and protein of Erk 1 increased between 7 and 28 days after axotomy both in the vagal and hypoglossal nuclei, however, there was no remarkable change in those of Erk 2. The increased expression of Erk 1 is common to both regenerative hypoglossal and degenerative vagal neurons. These findings indicate that Erk 1 is closely related with the retrograde neuronal reaction but whether neurons are destined to survive or die depends on some other factors.

Pallido-nigro-luysian atrophy, progressive supranuclear palsy and adult onset Hallervorden-Spatz disease: a case of akinesia as a predominant feature of parkinsonism.

Few parkinsonian patients present with 'pure akinesia' or with severe akinesia accompanied by only mild rigidity, tremor and other manifestations such as ophthalmoplegia. Pathological examinations of such cases have rarely been conducted and have revealed findings compatible with progressive supranuclear palsy (PSP), pallido-nigro-luysian atrophy (PNLA) or Parkinson's disease. We report a parkinsonian patient whose main clinical feature was akinesia. A postmortem study of this patient showed findings corresponding to PNLA and PSP. Histochemical properties of the pallidal pigment granules were equivalent to those of Hallervorden-Spatz disease (HSD) and striatonigral degeneration. In addition to iron-positive pigment granules, spheroids, severe neuronal loss and gliosis in the globus pallidus and substantia nigra, formation of Alzheimer's neurofibrillary tangle (NFT) in the brainstem shares characteristics with PSP, adult onset HSD and PNLA. We suggest that the underlying pathology of 'pure' akinesia is most often situated in the globus pallidus substantia nigra and subthalamus (Luys), and that PSP, PNLA and adult onset HSD may constitute a spectrum of one disease.

Epstein-Barr virus in the proliferative diseases of squamous epithelium in the oral cavity.

The presence of Epstein-Barr virus was analyzed in 79 cases of oral epithelial proliferative diseases by polymerase chain reaction, in situ hybridization for Epstein-Barr virus-deoxyribonucleic acid and Epstein-Barr virus-encoded small messenger ribonucleic acid and immunohistochemistry for Epstein-Barr virus latent membrane protein. These lesions were histologically categorized as invasive squamous cell carcinoma (n = 36), carcinoma in situ (n = 10), verrucous carcinoma (n = 4), leukoplakia (n = 19), and papilloma (n = 10). Epstein-Barr virus genomes were detected in 19 squamous cell carcinoma (52.8%), four carcinoma in situ (40%), and one leukoplakia (5.3%); none of the verrucous carcinoma or papilloma cases were positive with polymerase chain reaction. By deoxyribonucleic acid in situ hybridization, positive signals were observed in the nuclei of cancer cells in 10 cases, in infiltrating lymphocytes in three, and both in one case. In patients with carcinoma in situ, only a single case was positive. In one case of leukoplakia positive signals were found in upper and middle layer squamous cells. The results by Epstein-Barr virus-encoded small messenger ribonucleic acid in situ hybridization revealed the same distribution as that by deoxyribonucleic acid in situ hybridization. Latent membrane protein was expressed only in the epithelial cells of leukoplakia but not in cases with squamous cell carcinoma and carcinoma in situ. These findings suggest that Epstein-Barr virus infection of oral squamous epithelium may be carcinogenic; alternatively, the virus may merely exist in epithelial cells of squamous cell carcinoma, carcinoma in situ, and leukoplakia as a passenger.

Self-setting hydroxyapatite cement as a carrier for bone-forming cells.

To investigate the feasibility of using self-setting hydroxyapatite cement as a carrier for marrow cells having a high osteogenic ability, a porous form of this cement was fabricated and combined with cultured marrow cells. Marrow cells were obtained from the femurs of a seven-week-old male Fischer 344 rat and cultured in Eagle's MEM containing 15% fetal bovine serum for ten days before being combined with the porous cement or with Interpore 200 hydroxyapatite as a control. The composites were subcutaneously implanted into syngeneic rats and harvested after six weeks. In both types of implants, active osteoblasts together with bone formation were detected in contact with the pore surfaces. No cartilage formation was observed in any of the pores. Both types of implants with and without marrow cells caused very little foreign body reaction. These results indicate that self-setting hydroxyapatite cement containing marrow cells possesses a high osteogenic ability and may be useful as a bone graft substitute as well as a novel delivery system for bone-forming cells.

Human marrow cells-derived cultured bone in porous ceramics.

From four patients (mean age, 60 years; range 51-76 years), 3 ml of bone marrow was collected from the ilium. The marrow was cultured to concentrate and expand the marrow mesenchymal cells on a culture dish. The cultured cells were then subcultured either on another culture dish or in porous areas of hydroxyapatite ceramics in the presence of dexamethasone and beta-glycerophosphate (osteogenic medium). The subcultured tissues on the dishes were analyzed by scanning electron microscopy (SEM), and subcultured tissues in the ceramics were implanted intraperitoneally into athymic nude mice. Vigorous growth of spindle-shaped cells and a marked formation of bone matrix beneath the cell layers was observed on the subculture dishes by SEM. The intraperitoneally implanted ceramics with cultured tissues revealed thick layer of lamellar bone together with active osteoblasts lining in many pore areas of the ceramics after 2 months. The in vitro bone formation on the culture dishes and in vivo bone formation in porous ceramics were detected in all cases. These results indicate that we can assemble an in vitro bone/ceramic construct, and due to the porous framework of the ceramic, the construct has osteogenic potential similar to that of autologous cancellous bone. A significant benefit of this method is that the construct can be made with only a small amount of aspirated marrow cells from aged patients with little host morbidity.

[A case of intracranial germinoma with multiple remote metastases (author's transl)].

A case of suprasellar germinoma with multiple remote metastases was reported including its autopsy findings. Several series of radiation therapy and prolonged steroid administration were considered to be causes of immunosuppression bringing about such metastases. A 29-year-old man had undergone a suprasellar germinoma removal and 5,200 rad of postoperative local irradiation at a university hospital 5 months prior to the admission to our clinic. A walnut-sized left cerebellopontine angle mass was subtotally extirpated by us, which proved to be germinoma. Total dosis of 5,5550 rad with lineac was applied locally to the operated site. Since the second surgery, several nodules had been seen in the lumbosacral region, scalp and left cervical region, which were individually treated with lineac and beta-tron by 5,000, 6,000 and 4,750 rad. The patient had been on steroid substitution therapy for hypopituitarism since the first operation. Eventually the patient expired 9 months after the second operation. Autopsy findings included multiple metastases to the spinal arachnoid, dura, extradural space, spines, scalp, thoracic cage, lymph nodes, heart, lungs, liver, kidney, adrenal and pancreas. Nine cases including ours were reviewed as remote metastases of the tumors in the pineal region and of so-called ectopic pinealomas. The cases of only subarachnoid seeding of these were excluded in this study.

[A case of so-called carcinosarcoma of the lung].

A 65-year-old male was admitted to our institute because of bloody sputum. A tumor in right S6 was detected by X-ray, CT and MRI. Bronchoscopic study showed that the right lower bronchus was occluded by the tumor, in which non-epithelial malignant cells were detected. Therefore right bilobectomy was performed. This tumor was a pedunculated endobronchial type measuring 6 x 4 x 3cm. Histologically, the tumor presented carcinomatous (squamous cell carcinoma and adenocarcinoma) and sarcomatous elements. Immunohistologically, many malignant cells were positively stained by vimentin and muscle-actin, which suggested differentiation from muscle components.

Breast cancer with reactive multinucleated giant cells: report of three cases.

We report here three cases of breast cancer with reactive multinucleated giant cells. The patients were among the 605 patients with breast cancer seen in the past 17 years at Tenri Hospital; the incidence of this variety of breast cancer was 0.5%. Enzyme histochemical and electron microscopic examination suggested that the giant cells were of histiocytic origin. However, results of immunohistochemical technique, S-100 protein, lysozyme, nonspecific cross-reacting antigen with carcinoembryonic antigen, alpha-1-antitrypsin, and alpha-1-antichymotrypsin, all currently used as markers of histiocytes, were negative. Because of the rarity of this variety of breast cancer, the biological significance of these unusual findings remains unknown.

Malignant chondroid syringoma. Report of a case with widespread metastasis and review of pertinent literature.

The authors report the autopsy case of a 73-year-old man with widespread, metastatic, malignant chondroid syringoma, with a long clinical history. Three years after resection of the primary tumor on the back, the tumor recurred at the same site, metastasized to the cervical lymph nodes 7 years later, and finally, after 13 years exhibited widespread metastases. Histologically, the tumor showed cords and nests of cuboidal or polygonal tumor cells with little cellular pleomorphism and few mitoses surrounded by a mucoid matrix. Histochemically, the matrix contained hyaluronic acid and sulfated acid mucopolysaccharides, such as chondroitin sulfate A and/or C. To the knowledge of the authors there have been nine reported cases of malignant chondroid syringoma in the English literature, four of which had distant metastasis. A review of the literature reveals that malignant chondroid syringoma is more common in women and occurs most often in trunk and extremities, which is in contrast to its benign counterpart, its histologic appearances vary greatly, and it may even have benign appearances.

An unusual case of metaphyseal chondrodysplasia with an abnormal perilacunar matrix associated with agranulocytosis and hypoplasia of the thymus.

We report herein an unusual skeletal dysplasia in a 6-month-old boy characterized by metaphyseal dysplasia associated with agranulocytosis and hypoplasia of the thymus. A radiological survey revealed generalized metaphyseal abnormalities showing widening and increased density. Pathological changes in the chondro-osseous tissues were unusual and distinctive. There was widespread evidence of abnormal chondrocytes with an abnormal perilacunar matrix containing a PAS-positive, diastase resistant substance. Chondrocyte maturation and regular columnar arrangement were absent in most growth plates with only scattered ball-like nests of chrondrocytes showing incomplete maturation. This case is a newly described form of osteochondrodysplasia.