Estrogen reduces mechanical injury-related cell death and proteoglycan degradation in mature articular cartilage independent of the presence of the superficial zone tissue.

OBJECTIVE: To study the effect of 17ß-estradiol (E2) and the superficial zone (SFZ) on cell death and proteoglycan degradation in articular cartilage after a single injurious compression in vitro. METHOD: Cartilage explants from the femoropatellar groove of 2 year old cows with or without the SFZ were cultured serum-free with physiological concentrations of E2 and injured by an unconfined single load compression (strain 50%, velocity 2 mm/s). After 96 h cell death was measured histomorphometrically (nuclear blebbing (NB) and TUNEL staining) and release of glycosaminoglycans (GAG) by DMMB assay. RESULTS: Injurious compression increased significantly the number of cells with NB and TUNEL staining and release of GAG. Physiological concentrations of E2 prevented the injury-related cell death and reduced the GAG release significantly in a receptor-mediated manner (shown by co-stimulation with the antiestrogen fulvestrant/faslodex/ICI-182,780). The presence of the SFZ did not alter the NB response to either the mechanical injury or E2, but reduced the overall release of GAG significantly. CONCLUSION: E2 prevents injury-related cell death and GAG release, and might be useful for the development of treatment options for either cartilage-related sports injuries or osteoarthritis (OA). The SFZ does not seem to play an important role in (1) the E2-related tissue response and (2) the mechanically-induced cell death in deeper regions of the explants and GAG release. The latter might be related to the unconfined nature of the injury model.

Tumor risk by tissue engineering: cartilaginous differentiation of mesenchymal stem cells reduces tumor growth.

OBJECTIVE: Implantation of autologous chondrocytes (AC) is a promising option for the treatment of cartilage defects, but problems with cell harvesting, dedifferentiation, or the donor age limit the clinical outcome. Mesenchymal stem cells (MSC) gain much interest because of their simple isolation and multipotential differentiation capacity along with their immunosuppressive properties. The latter might introduce tumor manifestation. The influence of undifferentiated and chondrogenically differentiated MSC or AC on tumor growth and metastasis formation was investigated in a murine melanoma model. METHODS: Allogeneic melanoma cells and either syngeneic MSC (C3H10T1/2, transduced with enhanced green fluorescent protein gene) or AC were co-injected at a distance of 3 cm into the contra lateral groins of five mice/group, and evaluated macroscopically and histologically after 4 weeks. RESULTS: Undifferentiated MSC migrated to the tumor site and induced strong tumor growth and metastasis formation. Even avital MSC promoted tumor growth and spreading, but insignificantly without detectable MSC at the tumor site. Chondrogenically differentiated MSC did not migrate and had a significantly lower impact on tumor growth and spreading; AC had no measurable influence on melanoma cells. CONCLUSIONS: Our data suggest that differentiation of MSC reduces MSC-dependent promotion of latent tumors and that native AC do not introduce any increased risk of tumor growth. The question of how far MSC should be differentiated prior to clinical application should be addressed in further studies.

Beta irradiation decreases collagen type II synthesis and increases nitric oxide production and cell death in articular chondrocytes.

BACKGROUND: When synovitis is proved, intra-articularly injected beta emitting radionuclides like yttrium-90 ((90)Y) are used to treat the inflamed synovium. OBJECTIVE: To study the viability, matrix production, and NO production during or after (90)Y treatment of chondrocytes. METHODS: Monolayer, alginate, and explant cultures of primary bovine articular chondrocytes as well as synoviocytes were incubated with 0-3 MBq (90)Y/ml medium for four days from culture day 3 onwards. Cell viability was demonstrated by light and electron microscopy or by trypan blue or ethidium bromide/fluorescein diacetate staining, membrane integrity by measurement of lactate dehydrogenase (LDH) activity in the culture supernatants. Biosynthetic activity was demonstrated by incorporation of [(3)H]proline and immunocytochemical staining of collagen type II. NO production was measured with the Griess reagent. RESULTS: In chondrocyte and synoviocyte monolayer cultures radiation caused a dose dependent increase in cell death and membrane destruction within four days. In alginate and explant cultures, where proliferation is low, no significantly increased LDH activity was seen, and cell viability was approximately 100% for up to 14 days after irradiation. Collagen type II expression (alginate) and biosynthetic activity (alginate and explants) were decreased dose dependently while there was an increase in NO production. Light and electron microscopy data showed that five weeks after irradiation all cells in alginate and most cells in explants subjected to 3 MBq (90)Y/ml were dead, whereas after lower amounts of irradiation several morphologically intact cells were found. CONCLUSIONS: beta Irradiation may influence the long term maintenance of cartilage tissue or the aetiology of degenerative joint diseases.

Immunohistochemical detection of estrogen receptor alpha in articular chondrocytes from cows, pigs and humans: in situ and in vitro results.

Clinical observations suggest that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis, but only little is known about the influence of these hormones on articular cartilage cells. The effect of estradiol is mediated by estrogen receptors alpha and beta. The goal of the present study was to search for estrogen receptor alpha in articular tissue from cows, pigs and humans by immunohistochemistry to form a basis for in vitro studies. In addition, we also tried to detect estrogen receptor alpha in cultivated articular chondrocytes from cows and bulls under certain culture conditions. Estrogen receptor alpha is detected by the use of antibody 13H2 in articular chondrocytes from cows, bulls, pigs and humans. Chondrocytes are physiologically exposed to reduced oxygen tension. In isolated articular chondrocytes from cows and bulls incubated either with 21% O2 or with 5% O2 positive cells were also found. These positive results therefore encourage testing the influence of estradiol on cultivated articular cartilage cells in these species under different culture conditions.

Magnetic resonance arthrography of the acetabular labrum. Macroscopic and histological correlation in 20 cadavers.

We studied the sensitivity and specificity of magnetic resonance arthrography (MRa) for the diagnosis of lesions of the acetabular labrum in 20 cadaver hips. The MRa results were compared with macroscopic and histological findings. We found that the labrum could be satisfactorily delineated by MRa and that large detachments could be identified satisfactorily. The diagnosis of small detachments and degeneration of the labrum was less reliable.

Articular chondrocytes and synoviocytes in culture: influence of antioxidants on lipid peroxidation and proliferation.

Chondrocytes and synoviocytes are the main cell types in articular joints. Articular cartilage is fed by synoviocytes via synovial fluid and has a low partial oxygen pressure. Thus, chondrocytes show oxygen radical protective mechanisms in vivo and are unprotected against these factors under common culture conditions. We investigated the influence of ascorbic acid, Fe2+, glutathione and alpha-tocopherol on lipid peroxidation and proliferation of rat articular chondrocytes and rabbit synoviocytes (HIG-82) in vitro. A combination of ascorbic acid and Fe2+ induced the production of thiobarbituric acid-reactive material as a marker of radical-mediated lipid peroxidation in homogenates and/or supernatants of cultured chondrocytes and synoviocytes. The amount of lipid peroxidation of chondrocytes was about 3-fold higher than that of synoviocytes. Ascorbic acid or Fe2+ alone had no significant influence on the production of thiobarbituric acid-reactive material. Lipid peroxidation could be abolished by addition of the radical scavenger alpha-tocopherol, whereas glutathione had no effect. 25-50 microM alpha-tocopherol decreased the ascorbic acid-(100 micrograms/ml) and Fe(2+)-(3 microM) induced lipid peroxidation to a basal level. Moreover, ascorbic acid inhibited the proliferation of rat chondrocytes and rabbit synoviocytes measured by [3H]-thymidine incorporation. Alpha-tocopherol and glutathione had no influence on the proliferation of chondrocytes but alpha-tocopherol decreased the growth of synoviocytes and increased the anti-proliferative effect of ascorbic acid on these cells. The importance of these findings for the use of ascorbic acid, glutathione and alpha-tocopherol in chondrocyte and synoviocyte cultures, or the influence of these molecules on the etiology and treatment of articular diseases will be discussed.

In vivo monitoring of splenocytes in NMRI nu/nu- and nu/+,- mice during Lewis lung tumor progression or regression.

Splenocytes of T-cell deficient NMRI nu/nu- mice (n=15) and of inbred immunocompetent NMRI nu/+,littermates (n=18) significantly (p<0.05) increased during Lewis lung (LL) tumor growth and eventually decreased. Half of the heterozygous NMRI nu/+,- mice rejected the tumors (LL-regressors). Zymosan-induced and lucigenin-amplified chemiluminescence (CL) of splenic polymorphonuclear leukocytes (PMN) and -precursors significantly (p<0.05) increased in NMRI nu/+,- mice with progressing tumors (LL-progressors) on the 3rd and in LL-regressors and nude mice on the 7th day after tumor inoculation. It subsequently decreased although numbers of PMN/- precursors further multiplied predominantly in the LL-progressors. Another peak of CL was found in those animals after the 20th day. Splenic lymphocytes significantly (p<0.05) increased in all mice from day 5 forward and dropped to original numbers between the 20th and 25th days. Maximal lymphocyte counts were significantly (p<0.05) lower in nude mice as compared to the NMRI nu/+,- littermates. The splenic T-/B-ratio significantly (p<0.005) correlated in LL-regressors and did not in LL-progressors. This correlation could be a suitable marker for tumor progression in NMRI nu/+ mice bearing LL-tumors.

[Animal experiment studies of laser effects on lymphatic vessels. A contribution to the discussion of laser surgery segmental resection of carcinomas]. / Tierexperimentelle Untersuchungen zur Laserwirkung auf Lymphgefässe. Ein Beitrag zur Diskussion um die laserchirurgische Resektion von Karzinomen in mehreren Teilen.

BACKGROUND: The potential effect of laser surgery on the process of lymphogenic metastases is subject to controversy. In this context it would be of interest to identify a morphological basis for an increased intraoperative entry of tumor cells into the lymphatic system during resection of advanced carcinomas in two or more pieces via laser surgery. METHODS: The tissue effect of laser incisions (CO2 laser: 3030 W/cm2; Nd:YAG-laser: 3180 W/cm2) on the lymphatics of the buccal mucosa in 60 Wistar rats was studied by light microscopy, histochemistry, and lymphography during surgery and subsequent wound healing. These incisions were compared to conventional scalpel incisions. RESULTS: No lymphatic vessels were detected in the zone of carbonization, they were mostly dilated whereas in the zone of edema. In the zone of necrosis, the lymphatic were occluded by coagulation. The mid and long-term effect of the laser light on the regeneration of lymphatics was mainly correlated to wound healing which--when compared to conventional surgery--is delayed after laser surgery. Following scalpel incision, the first lymphatics enter the wound region after 4 days, a opposed to 10 days following laser surgery. Intact continuity of the lymphatic vasculature was detectable 15 days after scalpel incision and 42 days after laser incision. The regeneration of lymphatics was slightly more delayed after CO2 laser incision than after Nd:YAG laser incision. CONCLUSION: The results of this study do not indicate that the laser resection of carcinomas facilitates an intraoperative entry of tumor cells into the lymphatic vasculature. These experimental results verify clinical reports which do not observe an increased frequency of postoperative metastases after laser resection of carcinomas in two or more pieces.

[The laryngeal lymph vessel system of the human. A morphologic and lymphography study with clinical viewpoints]. / Das laryngeale Lymphgefässsystem des Menschen. Eine morphologische und lymphographische Untersuchung unter klinischen Gesichtspunkten.

Laryngeal carcinomas have different metastatic characteristics depending on their locations. Although these changes could be explained by differences in density of the lymphatic system, studies of the laryngeal lymphatic system have been controversial. To clarify these differences the lymphatic systems of 184 organ specimens were investigated with light and electron microscopy, histochemistry and injection techniques. Three types of lymphatic vessels were found in the larynx: lymph sinuses, precollectors and collectors. The highest density of lymphatics was found in the supraglottic larynx. From this region, lymph drained via 3-6 collectors to a hypopharyngeal collector. The subglottic lymph mainly drained via 2-4 collectors to paratracheal and mediastinal lymph nodes. The lymph system of the vocal folds was sparse but drained mainly to the supraglottic larynx. No lymph collector was found in the mucosa of the vocal fold. According to these results, the incidence of lymphatic spread correlated first with the density of the initial lymphatics in the various laryngeal regions and secondly (and probably of greater significance) with nearness of tumor to the laryngeal collectors.

[Origin of increased postoperative growth after laser surgical intervention in the peritoneal cavity--an experimental animal study]. / Zu den Ursachen vermehrter postoperativer Verwachsungen nach laserchirurgischen Eingriffen im Bauchraum--Eine tierexperimentelle Untersuchung.

This work deals with the localization and chronology of the appearance of tissue-plasminogen-activator (t-pa) in coagulated and vaporized peritoneal lesions. 21 female WISTAR-rats were laparotomized, similar areas of the parietal peritoneum being vaporized and coagulated. The excision of these lesions was done at seven preset time intervals from 0 to 48 hours postoperatively. The occurrence of t-pa and fibrin was shown by immunohistochemical staining of cryo- and plastic-embedded sections. The differentiation of cells was done with immuno- and enzymhistochemical techniques. The inflammatory response of vaporized lesions shows to be more intense, i.e. exsudation of more fibrin, higher amount of inflammatory cells and a strongly emphasized tendency of adhesion-formation. There is a very low amount of t-pa-reactive cells up to 12 hours postoperatively, increasing after 24 and 48 hours but staying low in relationship to the total amount of cells. In coagulated lesions there is a steady increase of t-pa-reactive cells up to 12 hours postoperatively. Adjacent undamaged peritoneal areas in both types of lesions show a significant t-pa-reactivity. The visible reactivity to t-pa can be localized almost exclusively around monocytes and peritoneal macrophages. Both types of lesions do contain closely the same number of KiM2R-reactive mononuclear cells. There is an increased number of mastcells in vaporized lesions. There is more research needed to be done on the influence of different traumatic impacts on the peritoneum in order to find a causal approach to prevention of adhesions. Right now the only causal way to minimize the incidence of iatrogenic postoperative adhesions is the use of the Semm's well known Endo-coagulation-Technique.

Morphological studies on the pathogenesis of Reinke's edema.

Light microscopy of vocal cord mucosa in patients with Reinke's edema revealed highly ramified fissured spaces in the subepithelial tissue that were generally lined with flat cells. The ultrastructure of the parietal cells resembled fibroblasts whose cytoplasmic extensions overlapped in two to three layers in some places. Cell contacts were not observed. Neither electron microscopy nor immunohistochemical testing with antibody against laminin demonstrated a basal membrane. It was possible to distinguish between light and dark cells in the specimens examined. The cytoplasm of the light cells contained intermediate filaments, mitochondria, lysosomes, coated vesicles, caveolae and broad cisternae of rough endoplasmic reticulum. The dark cells were more numerous and typically exhibited a well-developed endoplasmic reticulum and free ribosomes. The parietal cells showed no immunoreaction against human vascular endothelial cells. Immunohistochemical demonstration of mesenchymal intermediate filaments using antibody against vimentin yielded a positive reaction for some of the cells in the walls of the crevices and subepithelial tissue. It was also possible to demonstrate a few cells with monoclonal antibody against macrophages (KiM6). These findings contradict the concept of lymphatic distension in cases of Reinke's edema. Since the parietal cells seen resembled synoviocytes in their structure and immunohistochemical reactions, findings indicate that the hollow spaces of Reinke's edema develop like neobursae from mechanical strain.

Incidence of adhesions following thermal tissue damage.

Coagulation and vaporization of tissue are techniques applied in pelviscopic surgery in order to achieve hemostasis as well as cut and destroy endometriotic implants. An animal experimental study was devised to show if there is a difference in the incidence of adhesions after vaporization of equal-sized areas of the anterior abdominal wall of the rat compared to coagulation of equal-sized areas. A similar depth of the lesions was achieved by repeating the vaporization procedure. The rate of adhesions was significantly lower (P less than 0.001, Chi2-test) post-coagulation, using a biopolar high frequency current or post-endocoagulation than post-vaporization. The surface vaporization of tissue, for example endometriotic implants, as produced by laser is to be viewed critically as regards the higher incidence of adhesions after vaporization compared with coagulation.

Width of thermal damage after using the YAG contact laser for cutting biological tissue: animal experimental investigation.

At the University Women's Clinic in Kiel, the YAG contact laser has been used as a cutting instrument in pelviscopic operations since 1987. When the laser cuts, it produces only a scant amount of mechanical trauma. The determining factor is the amount of thermal damage produced along the wound margins and in direct neighboring tissue. The extent of the tissue change seen in the uterus and liver parenchyma of rats and the striated muscle of rabbits after application of the YAG contact laser was demonstrated using various staining techniques and stains. Liver parenchyma proved to be the most sensitive to thermal damage. In the uterine horn, enzyme-histochemical ATPase and alkaline phosphatase demonstrations showed a significantly wider zone of thermal damage after laser incision than did hematoxylin-eosin and Goldner staining techniques. A good understanding of the extent of thermal damage is essential for atraumatic pelviscopic operations using the YAG contact laser and also for the preventing of complications.

Description and clinical importance of the lymphatics of the vocal fold.

Findings of the presence of lymphatics in the subepithelial connective tissue layer of the true vocal cord range from the statement that lymphatics are absent to the observation that a dense lymphatic network exists. This is undoubtedly a result of the different methods of examination. The entire extent of a lymphatic system can be shown by either an enzyme histochemical demonstration of the 5'-nucleotidase activity in the lymphatics, or electron microscopy, which is more elaborate. These two methods are used to describe a subepithelial lymphatic network of varying density in 80 human vocal folds, which--contrary a frequent assumption--is not interrupted in the area of the free margin of the true vocal cord. Under the squamous epithelium, the density of the lymphatic system, which is very high in the arytenoid region, decreases continuously toward the anterior portion of the true vocal cord. It is exactly in the region in which the lymphatic system is the sparsest that almost all cancers of the true vocal cord develop. This finding is highly significant, in view of the low incidence of lymphogenous metastasis formation in T1-glottic cancers.

[Distribution of lymph vessels in the plica vocalis of the human. A light microscopy, enzyme histochemistry and electron microscopy study]. / Verteilung der Lymphagefässe in der Plica vocalis des Menschen. Eine lichtmikroskopische, enzymhistochemische und elektronenmikroskopische Untersuchung.

Results of studies on the presence of lymphatic capillaries in the plica vocalis are shown, using improved methods of preparation. Immediately after laryngectomy, followed by immediate fixation of the non-pathological plicae vocalis, large lymphatic capillaries can be easily identified in the specimen. Lymphatic capillaries, which cannot be seen in conventional light microscopy, are now histochemically demonstrated with a marker of 5' nucleotidase. These histochemical results are confirmed by electron microscopy. Using a combination of both methods, a different quantity of subepithelial lymphatic capillaries can be visualised over the whole length and width of the plica vocalis. Lymphatic capillaries below the stratified squamous epithelium in the region of the anterior commissure grow gradually more numerous up to the folds of the arytenoid cartilage. Hence, in contrast to previous studies, the superficial lymphatic capillary system of the laryngeal mucosae is not separated into a supraglottic and subglottic lymphatic net of the vocal cord.