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Preadipocyte seeded PLGA scaffolds for adipose tissue engineering.

Adipose tissue equivalents have not been addressed as yet despite the clinical need in congenital deformities, posttraumatic repair, cancer rehabilitation, and other soft tissue defects. Preadipocytes were successfully harvested from rat epididymal fat pads of Sprague-Dawley and Lewis rats and expanded ex vivo. In vitro cultures demonstrated full differentiation of preadipocytes into mature adipocytes with normal lipogenic activity. The onset of differentiation was well-controlled by regulating preadipocyte confluency. Poly(lactic-co-glycolic) acid (PLGA) polymer disks with 90% porosity, 2.5 mm thick, 12 mm diameter, pore size range of 135-633 microm were fabricated and seeded with preadipocytes at 10(5) cells/mL. Disks in vitro demonstrated fully differentiated mature adipocytes within the pores of the disks. Short-term in vivo experiments were conducted by implanting preseeded disks subcutaneously on the flanks of rats for 2 and 5 weeks. Histologic staining of harvested disks with osmium tetroxide (OsO4) revealed the formation of adipose tissue throughout the disks. Fluorescence labeling of preadipocytes confirmed that formed adipose tissue originated from seeded preadipocytes rather than from possible infiltrating perivascular tissue. This study demonstrates the potential of using primary preadipocytes as a cell source in cell-seeded polymer scaffolds for tissue engineering applications.

Adipocytes↗

Tissue microarrays: a current medical research tool.

Recent research in molecular biology has identified a significant number of novel markers, which may have diagnostic, prognostic and therapeutic significance. This is particularly pertinent in the field of cancer. Validation of these markers in multiple clinical specimens is currently performed by traditional histopathological techniques, which are disappointingly time consuming, labour intensive and, therefore, economically costly. These limitations have hampered the introduction of many novel markers into everyday clinical practice. The tissue microarray (TMA) is a high throughput technique, which allows the rapid and cost effective validation of novel markers in multiple pathological tissue specimens. Tissue from up to a 1000 histology blocks can be arrayed accurately onto a newly created paraffin block, at designated locations. Subsequently, morphological and molecular investigations can be performed to determine the clinical significance of the novel markers tested. It is now firmly established that the TMA can significantly accelerate the processing of a very large number of tissue specimens with excellent quality, good reliability and preservation of original tissue, with ultimate clinical benefit.

Biomarkers, Tumor↗

Recurrent hyperparathyroidism due to parathyroid autografts: incidence, presentation, and management.

Recurrent hyperparathyroidism (HPT) occurs in a small percentage of patients undergoing parathyroidectomy for primary HPT and is usually due to inadequate excision of hyperfunctioning parathyroid tissue in the neck, a missed ectopic and hyperplastic parathyroid, or, less commonly, parathyroid carcinoma and parathyroid autografts. In order to determine the incidence, clinical characteristics, and outcome of patients with recurrent HPT due to parathyroid autografts, we reviewed our experience with 604 consecutive patients operated on for primary HPT between 1965 and 1989. One hundred of these patients received parathyroid autografts consisting of portions of one or more parathyroid glands. Three patients with autografts, placed in the sternocleidomastoid muscle, developed recurrent HPT due to their autografts for an incidence of 3 per cent. Recurrent disease was diagnosed between 62 and 113 months with an average of 89 months. The autotransplants in all three of these patients were from hyperplastic or adenomatous parathyroid tissue. Two patients had a history of neck irradiation. Preoperative thallium scans accurately localized the hyperfunctioning parathyroid tissue in all three patients. At operation, the hyperfunctioning autografts had grown into a discrete mass with a single vascular pedicle and were resected. Histologic examination disclosed either hyperplastic or adenomatous tissue, and corresponded to the histology and location of the original tissue transplanted in each case. Follow-up ranges from 12 to 67 months, with an average of 48 months. All patients remain cured and none require oral calcium supplementation. We conclude that graft-dependent recurrent HPT is due to the autotransplantation of hyperplastic or adenomatous parathyroid tissue and that thallium scanning is instrumental for diagnosis and localization.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Incidentally detected giant oncocytoma arising in retroperitoneal heterotopic adrenal tissue.

A nonfunctional retroperitoneal oncocytoma incidentally discovered in a 40-year-old woman is described. The tumor, which was 17 cm in largest dimension, was completely separated from the kidneys and adrenal glands and consisted of nests of polygonal cells with large, granular, eosinophilic cytoplasm. Significant nuclear atypia, necrosis, and mitosis were absent. Ultrastructural analysis confirmed the oncocytic nature of the neoplastic cells. Since neoplastic cells were not immunoreactive for chromogranin and did not contain dense-core secretory granules, the diagnosis of oncocytic paraganglioma was excluded. Cells immunoreactive for 3beta-hydroxysteroid dehydrogenase, the enzyme catalyzing the conversions of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione, were identified in the tumor, thus strongly indicating adrenocortical tissue origin. Multiple nests of 3beta-hydroxysteroid dehydrogenase-positive cells were detected in the loose retroperitoneal connective tissue. These findings strongly support the origin of the tumor from heterotopic retroperitoneal rests of the adrenal gland. To our knowledge, only 1 similar case has been described in the literature to date.

3-Hydroxysteroid Dehydrogenases↗

Subtotal reconstruction of the nasal septum using a conchal reshaped graft.

The caudalmost section of the cartilaginous nasal septum performs the important function of supporting the middle and lower third of the nose. Its absence leads inevitably to deformation of the nasal pyramid and collapse of the internal nasal valve. The most frequent causes of its loss are iatrogenic and traumatic, and the mucoperichondrial lining remains intact in most cases. A graft of conchal cartilage constitutes the preferred method of reconstruction due to the capacity of the transplanted tissue to acquire characteristics of shape, elasticity, and strength closely resembling those of the original tissue to be replaced. The auricular concha differs in anatomic shape from the nasal septum and tends, when deformed, to return to its initial appearance due to cartilaginous memory. The auricular cartilage is also less robust than the quadrangular. The paper describes a surgical technique for reshaping of the conchal cartilage that makes it possible to obtain a practically straight conchal graft that will retain its stability over time with no risk of subsequent modification. The technique involves a double figure-of-8 suture together with incisions on the concave side of the graft for straightening purposes. The simultaneous use of 2 spreader grafts taken from and attached to the concha itself helps to maintain straightness and reinforce the structure. The graft is then placed in position via open access and secured between the 2 mucoperichondrial flaps after these have been carefully detached. The tissue and technique used make it possible to restore the original condition in anatomic and physiological terms, eliminating the esthetic impairment and regaining respiratory functionality.

Ear Cartilage↗

Persistence of herpes simplex virus genes in cells of neuronal origin.

The growth characteristics of the KOS strain of herpes simplex virus type 1 (HSV-1) in cell lines of nervous tissues origin were examined in an attempt to develop a tissue culture system mimicking the in vivo state of HSV-1 latency. We have previously reported that the B103 rat brain neuroma cell line is nonpermissive for growth of the KOS strain. In this report, we show that this nonpermissiveness is a temperature- and multiplicity-dependent phenomenon, with minimum virus yields at an elevated temperature and a low multiplicity of infection. Under these conditions, B103 cells survived infection with active wild-type or mutant HSV-1, whereas similarly treated Vero cells were killed. Six independent cultures of B103 cells surviving HSV-1 infection have been established. The surviving cells ceased production of any HSV-1 virus by 14 days postinfection and resumed growth and division at rates comparable to those of uninfected B103 cells. Survivor cells continued to express HSV-1-specific antigens, however, as detected by indirect immunofluorescence and by surface iodination followed by immunoprecipitation and polyacrylamide gel electrophoresis. The survivor cells did not express all of the surface proteins seen on productively infected B103 cells, and they were not susceptible to complement-mediated immune cytolysis with anti-HSV-1 antiserum. These results demonstrate that at least a portion of the HSV-1 genome is being harbored in these survivor cells.

Animals↗

Histologic and physiologic evaluation of skeletonized internal thoracic artery harvesting with an ultrasonic scalpel.

OBJECTIVES: The safety and reliability of a method of skeletonized internal thoracic artery harvesting with an ultrasonic scalpel (Harmonic Scalpel; Ethicon Endo-Surgery, CVG, Cincinnati, Ohio) were evaluated. METHODS: The mural branches of the internal thoracic artery were cut by means of 3 methods, differentiated by distance from the site of application of the Harmonic Scalpel blade to the internal thoracic artery. A total of 15 branches were cut from the internal thoracic artery at (0 mm) the origin (group I) or at 1 mm (group II) or 2 mm (group III) distal to the origin. Tissue preparations were examined for successful vessel closure and severity of tissue damage. The length of stump (L) and the length of tissue damage from the stump (D) were determined by a computer image analysis system, and pressure testing was performed to evaluate the physical strength of vessel closure. RESULTS: In group I, 8 of the 15 branches exhibited discontinuity of the vascular wall structure, probably because of insufficient sealing of the divided section, and 12 of the 15 branches exhibited tissue denaturation on the internal thoracic artery wall adjacent to areas of origin, which was probably caused by the heat transferred from the branches during the process of coagulation. In groups II and III, continuity of wall structure of stumps suggestive of stable closure of branches was confirmed. The lengths of tissue damage from the stump (D) were 0.96, 0.58, and 0.63 mm in groups I, II, and III, respectively, and the lengths of intact area (L - D) in the corresponding groups were -0.78, 0.61, and 1.51 mm. The negative figure in group I indicates the presence of tissue damage in the internal thoracic artery itself. By contrast, in groups II and III the internal thoracic arteries were intact, with a safety margin of greater than 0.5 mm. On physiologic evaluation of vessel closure, 2 of the 24 (8.3%) branches burst under a pressure lower than 350 mm Hg because of insufficient vessel coagulation, but the remaining 22 branches (91.7%) remained intact under pressures up to 350 mm Hg. CONCLUSION: The internal thoracic artery skeletonization method with an ultrasonic scalpel (Harmonic Scalpel: output level 2) appears to be a safe and reliable method of skeletonized internal thoracic artery harvesting when branches are sectioned at least 1 mm distal to their origin at a sufficiently slow speed.

Animals↗

The fate of the graft after extracorneal keratoplasty.

Two histological examinations are described. The first presents a full-thickness graft after extracorneal keratoplasty 2 years after transplantation. The graft is localized in the host tissue, externally lined by the thickened episclera and internally by the sclera. The reaction of the host tissue is slight and the graft is well preserved. In the second, a lamellar graft, rare capillaries grow 1 year after transplantation, the reaction of the host tissue is very mild, and the graft slowly changes into the original tissue of the host.

Adolescent↗

Extrahypothalamic luteinizing hormone-releasing factor (LRF): release of immunoreactive LRF in vitro.

The human placenta in vitro releases luteinizing hormone-releasing factor (LRF) which is immunologically indistinguishable from hypothalamic LRF. The release of LRF by placental tissue increased during the 4 days of culture and exceeded the original tissue content more than 5-fold. These data suggest that the human placenta may synthesize an LRF molecule immunologically identical with hypothalamic LRF.

Aging↗

Tissue microarrays and their relevance to the urologist.

PURPOSE: We review important aspects of TMA methodology and discuss its wide range of clinical applications with particular emphasis on key clinical studies. We also provide an update on recent and projected uses of this technology to help the urologist improve care in oncology patients. MATERIALS AND METHODS: A directed MEDLINE literature review of TMAs was performed. Important publications that have shaped our understanding of TMAs were selected for review. They were augmented by manual searches and our personal bibliographic collections. RESULTS: The TMA is a high throughput molecular biology technique that can significantly accelerate the processing of a large number of tissue specimens with excellent quality, good reliability and the preservation of original tissue. TMA studies demonstrate their accuracy and reliability compared to those of standard histological techniques and correlate with clinicopathological information to determine disease progression and prediction of the clinical outcome. CONCLUSIONS: This review represents an overview and update for the urologist on TMAs and their clinical applications in urological oncology. In the future it is anticipated that the outcomes of this method will be used to assist in the diagnosis, prognosis and development of novel therapies in individual patients.

Biomarkers, Tumor↗

Malignant schwannoma of the parapharyngeal space in von Recklinghausen's disease: a case report and review of the literature.

Neurofibromatosis (NF) or von Recklinghausen's disease is frequently accompanied by malignant tumours, which can occur at any site in the body. These malignancies are mainly of soft tissue origin. Of all head and neck malignancies, the number of soft tissue sarcomas is limited and in combination with NF this type of tumour is a rare event. In this report we describe the clinical course of a young female patient with NF, who presented with a massive malignant schwannoma in the parapharyngeal space, and review the pertinent literature.

Adolescent↗

A novel in vitro human model of hemangioma.

Hemangioma, the most common tumor of infancy, is characterized by a proliferation of capillary endothelial cells with multilamination of the basement membrane and accumulation of cellular elements, including mast cells. The initial rapid growth is followed by an inevitable but slow involution. The currently available therapies are empirical and unsatisfactory because what is known of the cellular and molecular basis of hemangioma development is rudimentary. Advances in the understanding of its programmed biologic behavior has been hampered by the lack of a valid human model. We report here a novel in vitro culture system that is a useful human model of hemangioma. A small fragment of hemangioma biopsy is embedded in fibrin gel in a well of culture plates and incubated in a serum-free, buffered-salt, minimal medium. A complex network of microvessels grows out from the tissue fragments. Biopsies taken from all three phases of hemangioma development were cultured successfully; proliferative phase samples developed microvessels in 1 to 4 days, involuting phase in 5 to 7 days, and involuted phase in 7 to 12 days. The relative growth rates of the microvessels in the culture of biopsies taken from different stages of hemangioma development reflect the growth patterns seen clinically. This model has been validated using histochemistry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. Comparison of the number, localization, and phenotype of endothelial and mast cells and the distribution of basement membrane constituents (type IV collagen, perlecan, and laminins) and growth factors (basic fibroblast growth factor, vascular endothelial growth factor, transforming growth factor-betas) in the biopsy and the tissue after culture shows that many of the characteristics of the original tissues were retained in culture. This in vitro human model of hemangioma overcomes some of the deficiencies associated with earlier models. It offers an opportunity for studying the precise cellular, biochemical, and molecular basis of hemangioma It may also help to elucidate the mechanisms of action of existing therapies and may lead to the identification of novel treatments for hemangioma.

Biomarkers, Tumor↗

[The interosseous palmaris muscle of the thumb. Results of an anatomic study of a muscle of the first ray].

In sixteen of twenty adult human cadaveric hands a muscle fascicle was isolated from the adductor pollicis muscle. It had osseous origins from the base of the first metacarpal bone and from the trapezium bone in eleven hands. These origins were connected by a fibrous arch, which was a soft tissue origin itself. In two of the sixteen hands, muscle fibers emerged from the palmar aspect of the first dorsal interosseous muscle near the radial artery. The findings can be explained by the ontogenesis of the human hand. The described muscle is neither a part of the oblique head of the adductor pollicis nor an equivalent to the three palmar interosseous muscles. In our dissections it was a constant muscle. Other studies have to show the exact function of the muscle. We postulate a stabilization of the carpometacarpal joint of the thumb.

Adult↗

Role of synthetic extracellular matrix in development of engineered dental pulp.

In cases of damaged oral tissues, traditional therapies, such as a root canal, replace the injured tissue with a synthetic material. However, while the materials currently used can offer structural replacement of the lost tissue, they are incapable of completely replacing the function of the original tissue, and often fail over time. This report describes a tissue engineering approach to dental pulp tissue replacement utilizing cultured cells seeded upon synthetic extracellular matrices. Human pulp fibroblasts were obtained and multiplied in culture. These cells were then seeded onto three different synthetic matrices: scaffolds fabricated from polyglycolic acid (PGA) fibers, a type I collagen hydrogel, and alginate in an effort to examine which matrix is most suitable for dental pulp tissue formation. In addition, methods previously developed for seeding and culturing pulp cells on PGA were optimized. Culturing cells on PGA resulted in a very high cell density tissue with significant collagen deposition. No cell proliferation was observed on alginate, and the growth of cells in collagen gels after 45 days was only moderate. These studies indicate dental pulp-like tissues can be engineered, and this may provide the first step to engineering a complete tooth.

Adult↗

Pancreatic tumours produce neurotensin.

Tumour tissue may secrete substances which are not normally secreted by the original tissue. We have found that 6 out of 21 pancreatic tumours producing vasoactive intestinal peptide also produce neurotensin-like peptides. These are sometimes secreted and very high plasma levels of neurotensin-like immunoreactivity may be found in the circulation.

Female↗

Virulence and nucleotide sequence analysis of marine viral haemorrhagic septicaemia virus following in vivo passage in rainbow trout Onchorhynchus mykiss.

A marine isolate of viral haemorrhagic septicaemia virus (VHSV) (860/94) was passaged in triplicate through sequential batches of rainbow trout via an intra-peritoneal infection route, without amplification in tissue culture. Following 5 passages, the VHSV glycoprotein gene was amplified directly from fish tissue homogenates and the consensus sequence compared to that of the original tissue culture isolate. Virus was also recovered directly from pools of kidney and spleen material after 5 passage events, and its virulence compared to that of unpassaged material by intra-peritoneal infection. Following passage in rainbow trout, isolate 860/94 exhibited a higher virulence for rainbow trout than unpassaged material. Sequence comparisons identified no difference in the consensus sequence of the glycoprotein gene following in vivo passage. The mechanisms responsible for the observed increase in virulence of isolate 860/94 following passage in rainbow trout thus remain unknown. The possibility that viral isolates may exhibit an increased virulence following passage in novel host species does, however, have important implications with regard to the epidemiology of this important fish pathogen.

Animals↗

Cutaneous changes during prolonged application of 12-O-tetradecanoylphorbol-13-acetate on mouse skin and residual effects after cessation of treatment.

The epidermal and dermal effects of protracted 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment (2 micrograms TPA twice weekly) of Sencar mouse skin were studied using cell kinetics and morphometric techniques. In addition, regression of TPA-induced changes was evaluated after cessation of 56 topical applications. During the first week of treatment a reactional hyperplasia, characterized by cell damage, edema, and acute inflammation in both epidermis and dermis, occurred. This picture changed gradually during the following 3 weeks: an epidermal hyperplasia devoid of involutional or inflammatory features was accompanied by a moderate to mild chronic inflammation of the dermis and a hyperplasia of the hair follicles. This remained throughout the experimental period until the topical TPA treatment ceased. Although TPA induced papillomas in only 5% of the animals (maximum = 2 papillomas/animal and no carcinomas), all sustained marked epidermal hyperplasia of approximately 4 to 5 times the normal thickness, and increased the number and volume of hair follicles. The [3H]thymidine pulse-labeling index of the basal layer was approximately 32% (normal congruent to 6%). The level of dark keratinocytes remained constant; i.e., 8% of the basal cells were identified as dark cells during the entire experiment. At the subepidermal level the dermal thickness and total cellularity increased, although the proportion of the different cell types changed during the treatment. The mast cell population increased remarkably. After TPA treatment ceased, most of these parameters regressed abruptly during the first 2 weeks. Two to 4 months later, the epidermis was slightly thinner, and the labeling index was 50% lower than normal (2.8%). This study shows that prolonged repetitive TPA applications induced a steady-state hyperplasia without tachyphylaxis, and that this alteration regressed rapidly after treatment ceased. In addition, labeling-index values lower than normal were reached soon after normalization, suggesting that a possible selection of keratinocytes, dependent on TPA for proliferation, took place during the chronic administration of topical TPA. The number of hair follicle, capillary vessels, mast cells, and the dermal thickness never reached normal values after treatment. These important changes in the dermis and hair follicles indicate that the target cells for tumor promoters are not confined to the epidermis alone, and that these tissues could participate actively in carcinogenesis directly, either as tumor-originating tissues (hair follicles), or as inducers or helpers of neoplastic growth (connective tissue cells).

Animals↗

A survey on neoplasia in domestic species over a 40-year period from 1935 to 1974 in the Republic of South Africa. II. Tumours occurring in sheep.

A survey was carried out on all ovine neoplasms recorded in the registration files of the Section of Pathology of the Veterinary Research Institute at Onderstepoort over a 40-year period from 1935 to 1974. The neoplasms were divided and tabulated into 8 groups according to body systems or tissue types. Out of a total of 673 neoplasms, 436 (64,8%) were cases of jaagsiekte (pulmonary adenomatosis). Of the remaining 237 neoplasms, 41,3% involved the skin. Eighty per cent of the cutaneous neoplasms were squamous cell carcinomas which varied from well-differentiated to anaplastic. The majority occurred on the head, in particular on the ears, frontal region and on the eyelids or nictitating membrane. Several factors have been suggested to explain the high incidence of squamous cell carcinomas on the head. Lymphosarcomas were the 3rd most commonly encountered tumours, whilst a significant number of hepatocellular carcinomas also occurred. A variety of tumours of connective tissue origin were recorded, the most common of which arose from fibrous tissue and cartilage.

Animals↗