Absence of lymphatic vessels in human dental pulp: a morphological study.

Few and controversial data are available in the literature regarding the presence of lymphatic vessels in the human dental pulp. The present study was designed to examine morphologically the existence of a lymph drainage system in human dental pulp. Human dental pulp and skin sections were immunohistochemically stained with specific antibodies for lymphatic endothelium (D2-40, LYVE-1, VEGFR-3 [vascular endothelial growth factor receptor-3], and Prox-1), with the pan-endothelial markers CD31 and von Willebrand factor (vWF), and with the blood-specific marker CD34. Several blood vessels were identified in human pulps and skin. Lymphatic vessels were found in all human skin samples but in none of the pulps examined. Western blotting performed on human dermis and on pulps treated with collagenase (to remove odontoblasts) confirmed these results. Transmission electron microscopy indicated that vessels which, by light microscopy, appeared to be initial lymphatic vessels had no anchoring filaments or discontinuous basement membrane, both of which are typical ultrastructural characteristics of lymphatic vessels. These results suggest that under normal conditions human dental pulp does not contain true lymphatic vessels. The various theories about dental pulp interstitial fluid circulation should be revised accordingly.

The topography of microstructured surfaces differently affects fibrillin deposition by blood and lymphatic endothelial cells in culture.

While tissue-engineered blood vessels have already been successfully used in surgical practice, artificially restoring lymphatic circulation when needed is still far to be realized. Stability of arterial vessel wall depends on proper fibrillin deposition; fibrillin in fact is the scaffold for elastic fiber formation. In lymphatic vessels fibrillin is probably implied in lymph formation in response to interstitial requirements. This study was designed to verify whether fibrillin deposition is influenced by the topography of the substrate on which blood and lymphatic endothelial cells grow. Blood and lymphatic endothelial cells were cultured on microstructured surfaces with different topography: stripes of different widths (25, 50, and 100 microm), squares and rectangles, and spiral geometry, obtained by the photoimmobilization of Hyaluronan (Hyal) on aminosilanized glass. Cell orientation and fibrillin deposition were influenced by the topography of the microstructure. Blood endothelial cells deposited fibrillin as a bundle running parallel to the major axis of stripes and spirals, whereas the irregular network of fibrillin deposited by lymphatic endothelial cells was affected by the topography of the substrate only in the smallest stripes. These data bring a contribution to the basic knowledge required to design tissue-engineered blood and lymphatic vessels capable of adapting to the functional requirements of the surrounding environment.

Treatment of experimental injury of anal sphincters with primary surgical repair and injection of bone marrow-derived mesenchymal stem cells.

PURPOSE: Sphincter injury is a common cause of anal incontinence. Surgical repair remains the operation of choice; however, the outcome often is poor. We investigated the ability of injected bone marrow-derived mesenchymal stem cells to enhance sphincter healing after injury and primary repair in a preclinical model. METHODS: Twenty-four inbred Wistar Furth rats were divided into three groups. As a control, Group A underwent sham operation. Group B had sphincterotomy and repair of both anal sphincters plus saline injections. The study group (Group C) underwent sphincterotomy and repair followed by intrasphincteric injections of syngenic bone marrow-derived mesenchymal stem cells. A further group (Group D) of outbred Wistar rats treated with mesenchymal stem cells and immunosuppressive therapy also was evaluated. At 30 days, histologic and morphometric analysis and in vitro contractility testing was performed. RESULTS: A significant decrease of muscle tissue was observed at the site of repair after sphincter injury. However, in Groups C and D, histologic examination demonstrated new muscle fibers and morphometric analysis revealed a significantly greater muscle area fraction than in Group B (P < 0.05). Moreover, mesenchymal stem cells injection improved contractility of sphincters strips compared with Group B (P < 0.05). No significant differences were found between Groups C and D. CONCLUSIONS: In our experimental model, bone marrow-derived mesenchymal stem cells injection improved muscle regeneration and increased contractile function of anal sphincters after injury and repair. Therefore, mesenchymal stem cells may represent an attractive tool for treating anal sphincter lesions in humans. Investigations into the biologic basis of this phenomenon should increase our knowledge on underlying mechanisms involved in sphincter repair.

Effect of anoxia-glucopenia and re-superfusion on intrinsic nerves of mammalian detrusor smooth muscle: importance of glucose metabolism.

AIMS: To investigate the effect of anoxia/glucopenia and re-superfusion on intrinsic nerves in the mammalian urinary bladder. METHODS: Strips of detrusor smooth muscle were dissected from monkey and human urinary bladder and mounted for tension recording in organ baths superfused with Krebs solution. Human, monkey, and guinea-pig urinary bladders were treated to evaluate glycogen contents by a biochemical method. RESULTS: Detrusor strips from both monkeys and humans had to be exposed to anoxia-glucopenia for up to 2-2.5 hr to observe a progressive decline in the response to electrical field stimulation (EFS) of the intrinsic nerves, at variance with guinea-pig detrusor strips. In contrast, the response to direct activation of the smooth muscle with carbachol remained almost unaltered. Incubation of human and monkey detrusor strips with 2-deoxyglucose (2-DG) during 1 hr anoxia-glucopenia, however, caused a marked damage to the intrinsic nerves. The glycogen contents of both human detrusor specimens and monkey urinary bladders were 2.0- and 1.4-fold higher, respectively, than that found in guinea-pig urinary bladder; furthermore, untreated monkey detrusor sections showed a greater number of glycogen granules as compared to those subjected to anoxia-glucopenia and re-superfusion. In guinea-pig and in monkey detrusor sections glycogen granules were found in smooth muscle cells but not in neurons of intramural ganglia. CONCLUSIONS: A higher susceptibility of guinea-pig as compared to monkey and human nerves has been demonstrated; it is suggested that anaerobic glucose metabolism during anoxia-glucopenia is crucial for the functional recovery of detrusor intrinsic nerves from damage caused by anoxia-glucopenia and re-superfusion.

Structure and molecular organisation of the sarcoplasmic reticulum of skeletal muscle fibers.

Activation of muscle contraction is a rapid event that is initiated by depolarization of the plasma membrane and transverse (T) tubules, which following transduction in the interior of the muscle cell, activate the release of calcium from the sarcoplasmic reticulum (SR). Pioneer studies using electron microscopy defined the organization of the sarcoplasmic reticulum and the details of the junctions between sarcoplasmic reticulum and T tubules, which are essential for translating the electrical signal on the plasma membrane to calcium release from the sarcoplasmic reticulum. Molecular biology and biochemistry studies have revealed the presence of several proteins located on the sarcoplasmic reticulum, some of which participate together with the ryanodine receptors to the assembly of a large multi-protein complex, while others, like the calcium pumps, have independent localization and activities. As a whole, the current view of this system contemplates the existence of a high level of structural organization in the sarcoplasmic reticulum with respect to the localization of ryanodine receptors and other proteins. In this review we shall summarize studies on the expression and possible functional significance of the ryanodine receptor type 3 in mammalian skeletal muscles and recent studies aimed to dissect the mechanisms that establish the organization of the SR in striated muscles.

Lymphatic vessels in colorectal cancer and their relation with inflammatory infiltrate.

PURPOSE: The aim of this study was to determine why colorectal tumors confined to submucosa rarely metastasize. Under normal conditions, the submucosa contains many large lymphatic vessels with thin walls that would presumably favor the spread of cancer cells through the lymphatic system. METHODS: Specimens of colorectal cancer tissue, the border between tumor and normal tissue, and normal tissue were obtained from patients undergoing radical resection of colorectal cancer. The material was embedded in methacrylate resin for light microscopy and Epon for transmission electron microscopy examination. Light microscopy observations were routinely performed on serial sections. RESULTS: No lymphatic vessels were ever found in the tumor mass. The border area contained peritumoral inflammatory infiltrate of variable thickness. Where submucosal lymphatic vessels came into contact with peritumoral inflammatory infiltrate, they were profoundly altered: their endothelium was fragmented, and their walls were disrupted. These altered lymphatic vessels were almost always accompanied by mast cells, which were observed in the process of degranulating toward the lymphatic endothelium. No such alterations were detected in blood vessels. CONCLUSION: Our results suggest that mast cells, probably influenced by inflammatory infiltrate and/or colorectal cancer cells, destroy lymphatic vessels, which prevents cancer cells from spreading through the lymphatic system.

Focal adhesion molecules expression and fibrillin deposition by lymphatic and blood vessel endothelial cells in culture.

The microfibrils of anchoring filaments, a typical ultrastructural feature of initial lymphatic vessels, consist mainly of fibrillin and are similar to the microfibrils of elastic fibers. As we previously demonstrated, they radiate from focal adhesions of lymphatic endothelium to the perivascular elastic network. Although present in large blood vessels, fibrillin microfibrils have never been detected in blood capillaries. Here we report immunohistochemical evidence that cultured bovine aortic and lymphatic endothelial cells express fibrillin microfibrils. These microfibrils form an irregular web in lymphatic endothelial cells, whereas in blood vessel endothelial cells they are arranged in a honeycomb pattern. Cultured lymphatic and blood vessel endothelial cells also produce focal adhesion molecules: focal adhesion kinase, vinculin, talin, and cytoskeletal beta-actin. Our data suggest that anchoring filaments of initial lymphatic vessels in vivo may be produced by endothelium. Through their connection with focal adhesions, they may form a mechanical anchorage for the thin wall of initial lymphatic vessels and a transduction device for mechanical signals from the extracellular matrix into biochemical signals in endothelial cells. The complex anchoring filaments-focal adhesions may control the permeability of lymphatic endothelium and finely adjust lymph formation to the physiological conditions of the extracellular matrix. The different deposition of fibrillin microfibrils in blood vessel endothelial cells may be related to the necessity of withstanding shear forces. Thus, in our opinion, differences in fibrillin deposition imply a different role of fibrillin in blood vessel and lymphatic endothelium.