Expression and Localization of aquaporin‐1 in Temporomandibular Joint Disc with Internal Derangement

Background: Internal derangement is the most frequent arthropathy affecting the temporomandibular joint, where its commonest form is anterior disc displacement with or without reduction. Despite the frequency of the disorder, the biochemical features of displaced discs are still unclear. Methods: We investigated the expression pattern and localization of aquaporin‐1, an important channel protein involved in plasma membrane water permeability, in patients with anterior disc displacement (both with and without reduction), with a view to assessing the characteristics of local tissue responses to the microenvironmental changes induced by abnormal mechanical loading of the displaced disc. Protein expression was studied by immunohistochemistry in different areas of discs from 18 patients with anterior disc displacement with or without reduction and in four normal controls. Results: A greater proportion of cells immunopositive for aquaporin‐1 were detected in diseased than in normal discs. Whereas protein expression was substantially similar in the different areas of normal discs, a significantly larger number of immunopositive cells were detected in the posterior band of displaced discs without reduction and in the anterior and intermediate bands of those with reduction. Conclusions: These findings suggest that aquaporin‐1 is expressed and upregulated in temporomandibular joint with anterior disc displacement (both with and without reduction)

Mesenchymal stem cells from adipose tissue differentiated in chondrocytes into three-dimensional clusters termed chondrocyte “nodules” express lubricin

Lubricin is recognized to have an important role in preventing cartilage wear and synovial cell adhesion and proliferation. This study focused on isolation, cultivation, characterization of mesenchymal stem cells (MSCs) from adipose tissue and on their differentiation into chondrocytes through the NH ChondroDiff Medium. The main aim was to investigate some biochemical markers, such as collagen type I and II and lubricin verify the possibility to suggest of employing autologous three-dimensional clusters termed chondrocyte “nodules” in cartilage repair. Three-dimensional chondrocyte “nodules” were assessed with histology (haematoxylin and eosin), histochemistry (Alcian blue and Safranin-O/fast green) and Hoechst 33258 staining. Collagen type I, II and lubricin expression was determined by immunohistochemistry, immunofluorescence and Western blot. The results showed that, compared to control cartilage and monolayer chondrocytes showing just collagen type I, chondrocytes from MSCs (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative) of adipose tissue grown in “nodules”, at 24 days, were able to express lubricin, collagen type I, and II, indicative of hyaline cartilage formation. Based on the function of lubricin in the joint cavity and disease and as a potential therapeutic agent, our results suggest the possibility of applying autologous cell transplantation from adipose tissue differentiated in chondrocyte “nodules”

Assessment of cryopreserved human tunica albuginea for the surgical treatment of penile defects

Peyronie’s disease, a connective tissue disorder of penile tunica albuginea (TA) associated with penile deformity, curvature, pain and erectile dysfunction, is best managed surgically, but suitable graft biomaterials are not available. To establish whether cryopreservation affects human TA in view of its use in allotransplants. The effects on TA samples of the two most widely used tissue cryopreservation methods were investigated using an ad hoc panel of histochemical, immunohistochemical and ultrastructural tests. Apoptotic cells were evaluated using the TUNEL assay. Assessment of tissue integrity and arrangement of collagen and elastic fibers in thawed TA. Both cryofixation methods provided TA tissue suitable for use as graft material. Significant ultrastructural changes, namely a greater diameter of collagen fibrils, were detected in sections preserved in liquid nitrogen; nonetheless, such increase never exceeded the normal range. The comprehensive panel of assays used proved suitable to characterize the thawed tissue. Human TA is suitable for cryopreservation; freezing at -80 °C provides better results than preservation in liquid nitrogen

Mechanically induced chondrogenesis of human bone marrow derived stem cells

Bone marrow derived mesenchymal stem cells (MSCs) have been shown to offer great promise in regenerating defects of the musculoskeletal system. Beside growth factors, mechanical load is known to regulate the phenotypic fate decision in these cells. So far, a number of studies have shown beneficial effects of load on chondrogenesis of MSCs. However, the proper mechanical input still remains unknown. This study aimed to compare the chondrogenic response of compression and shear on MSC differentiation, while varying the cell number on the upper-surface of the construct in the absence of exogenous chondrogenic stimuli. In this set of experiments, human MSCs will be seeded into fibrinpolyurethane (PU) composite scaffolds. Human bone marrow derived MSCs were seeded into three-dimensional PU scaffolds (4 x 8 mm) at a cell density of 4 x 106 per scaffold. Constructs were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) with no exogenous transforming growth factor-β (TGF-β). Scaffolds were exposed to 15 loading cycles over 3 weeks, thereby assigned to 4 groups: Group A was exposed to compression and shear 4 milions MSC cells seeded into the scaffold. Group B was the free-swelling 4 milions MSC cells seeded into the scaffold (unloaded) control. Group C was exposed to compression and shear 3.6 milions MSC cells seeded into the scaffold plus 400 thousands MSC cells seeded on the top. Group D was the free-swelling 3.6 milions MSC cells seeded into the scaffold plus 400 thousands MSC cells seeded on the top (unloaded) control. Measurements included DNA, glucosaminoglycan (GAG), Elisa analysis, histology, himmunohistochemistry and mechanical competence. In addition, mRNA expression of chondrogenic markers (collagen type-II (Col 2), Aggrecan (AGG), osteogenic markers (collagen type-I (Col 1), alkaline phosphatase (ALP) and hypertrophic markers (collagen type-X (Col 10)) were assessed. Mechanical load and shear led to an increase in all the chondrogenic genes investigated with the greatest effect in group C. On mRNA level, AGG and Col 2 were upregulated to a greater extend in loaded groups compared to no loaded groups. This was reflected in the histological analysis, where only the group C showed a matrix rich in glucosaminoglycan staining. These results highlight that Group C was exposed to compression and shear 3.6 milions MSC cells seeded into the scaffold plus 400 thousands MSC cells seeded on the top demonstrated to mechanically induce in vitro chondrogenesis of MSCs. This is important knowledge for the repair of musculoskeletal disorders, whereby the same cell type (MSC) can induce bone repair as well as cartilage repair depending on the mechanical cues. Furthermore, this insight provides valuable information to optimise rehabilitation procedures to be used after implantation of MSCs for cartilage repair

Dopamine D3 receptor, neurofibromin and amyloid precursor protein expression during learning

Among dopamine receptors, the Dopamine D3 receptor (D3R) has been extensively characterized. It is distributed with highest densities in the limbic system and plays an important role in cognitive, emotional and endocrine functions. Like D3R, NF1 and APP genes are also involved in memory processes. Neurofibromin is a large tumor suppressor protein encoded by Neurofibromatosis type I gene (NF1). Amyloid precursor protein (APP) is the precursor of the amyloid-beta (Aβ) peptides involved in the pathogenesis of Alzheimer’s disease. Previously, it has been proposed that neurofibromin forms a binding complex with APP that interacts with D3R (Donarum EA et al., 2006). In addition, we have demonstrated that the absence of D3R is correlated to modifications in the expression of both NF1 and APP. Since these genes are all involved in cognitive processes, we have investigated whether such correlation is also present during a specific learning task. D3R, NF1 and APP expression levels were assessed in hyppocampi of mice subjected to the passive avoidance test. Animals were divided into four groups: naive, unconditioned stimulus trained (USTA), conditioned stimulus trained (CSTA) and conditioned (CA). mRNA and protein levels were analyzed by quantitative real time PCR and Western blot. Results showed that hyppocampal D3R expression was significantly increased in CA as compared to naive and, to a greater extent, in CSTA and USTA. Concurrently, increased NF1 expression was also found in CA and CSTA, but not in USTA. APP expression was unchanged both in CA, in CSTA and USTA as compared to naive animals. In conclusion, these data suggest that D3Rs may be correlated to passive avoidance-related learning, whereas both NF1 and APP do not seem to be directly linked to D3Rs during the acquisition of this specific learning task

Dopamine D3 receptor modulates tissue type plasminogen activator (tPA) activity in mouse brain

Growing amount of evidence points to the dopamine D3 receptor (D3R) as an important mediator in the broad array of events that regulate memory function, perhaps through the modulation of molecular pathways involved in neurotrophic factor activation. Tissue type plasminogen activator (tPA) is a proteolytic enzyme that cleaves the precursor of brain derived neurotrophic factor (proBDNF) into the biologically active form of mature BDNF. However, whether D3Rs modulate tPA activity on BDNF in brain has not been ascertained yet. Here in the present study, using D3R knock-out (D3-/-) mice, we demonstrate that receptor inactivation is associated with increased tPA expression both in prefrontal cortex and, to a greater extent, in the hippocampus, two regions associated with memory processes. The heightened tPA levels observed in D3-/- mice inversely correlated with proBDNF protein expression, whereas they positively correlated with both BDNF mRNA and mature BDNF protein levels. In conclusion, our finding strongly suggest that D3Rs might modulate tPAmediated post-transcriptional processing of BDNF in brain regions critical to memory function

The effects of physical activity (treadmill and vibration stimulation training) on RANKL and OPG expression in bone cells, in rats with glucorticoid-induced osteoporosis

The aim of this study was to investigate bone tissue and plasma levels of RANKL and OPG in rats with prednisolone-induced osteoporosis and to evaluate the outcomes of physical activity on the skeletal system by treadmill and vibration platform training. Osteoporosis is a disease characterised by low bone mass and structural deterioration of bone tissue leading to bone fragility. Vibration exercise is a new and effective measure to prevent muscular atrophy and osteoporosis. The animals were divided into 5 groups. 1: control rats; 2: rats with osteoporosis receiving prednisolone; 3: rats receiving prednisolone and treadmill training; 4: rats receiving prednisolone and vibration stimulation training; 5: rats receiving prednisolone, treadmill training and vibration stimulation training. Bone evaluations used whole-body scans, histology and histomorphometric analysis. RANKL and OPG expression was evaluated by immunohistochemistry and biochemical analysis. After treatment, our data demonstrated that RANKL expression was significantly increased in groups 2 and 3 and decreased in groups 4 and 5. Conversely, OPG expression was significantly decreased in groups 2 and 3 and increased in groups 4 and 5. In conclusion, our findings suggest that mechanical stimulation inhibits the activity of RANKL. This finding provides new insights into the occurrence and progression of osteoporosis