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The malignity of lung cancer is conditioned by the tumor microenvironment (TME), in which cancer-associated fibroblasts (CAFs) are relevant. In this work, we generated organoids by combining A549 cells with CAFs and normal fibroblasts (NF) isolated from adenocarcinoma tumors. We optimized the conditions for their manufacture in a short time. We evaluated the morphology of organoids using confocal microscopy analysis of F-actin, vimentin and pankeratin. We determined the ultrastructure of the cells in the organoids via transmission electron microscopy and the expression of CDH1, CDH2 and VIM via RT-PCR. The addition of stromal cells induces the self-organization of the organoids, which acquired a bowl morphology, as well as their growth and the generation of cell processes. They also influenced the expression of genes related to epithelial mesenchymal transition (EMT). CAFs potentiated these changes. All cells acquired a characteristic secretory phenotype, with cohesive cells appearing inside the organoids. In the periphery, many cells acquired a migratory phenotype, especially in organoids that incorporated CAFs. The deposit of abundant extracellular matrix could also be observed. The results presented here reinforce the role of CAFs in the progression of lung tumors and could lay the foundation for a useful in vitro pharmacological model.

There is evidence that demonstrates the effect of cannabinoid agonists inhibiting relevant aspects in lung cancer, such as proliferation or epithelial-to-mesenchymal transition (EMT). Most of these studies are based on evidence observed in in vitro models developed on cancer cell lines. These studies do not consider the complexity of the tumor microenvironment (TME). One of the main components of the TME is cancer-associated fibroblasts (CAFs), cells that are relevant in the control of proliferation and metastasis in lung cancer. In this work, we evaluated the direct effects of two cannabinoid agonists, tetrahydrocannabinol (THC) and cannabidiol (CBD), used alone or in combination, on CAFs and non-tumor normal fibroblasts (NFs) isolated from adenocarcinoma or from healthy lung tissue from the same patients. We observed that these compounds decrease cell density in vitro and inhibit the increase in the relative expression of type 1 collagen (COL1A1) and fibroblast-specific protein 1 (FSP1) induced by transforming growth factor beta (TGFβ). On the other hand, we studied whether THC and CBD could modulate the interactions between CAFs or NFs and cancer cells. We conditioned the culture medium with stromal cells treated or not with THC and/or CBD and cultured A549 cells with them. We found that culture media conditioned with CAFs or NFs increased cell density, induced morphological changes consistent with EMT, inhibited cadherin-1 (CDH1) gene expression, and induced an increase in the relative expression of cadherin-2 (CDH2) and vimentin (VIM) genes in A549 cells. These changes were inhibited or decreased by THC and CBD administered alone or in combination. In another series of experiments, we conditioned culture media with A549 cells treated or not with THC and/or CBD, in the presence or absence of TGFβ. We observed that culture media conditioned with A549 in the presence of TGFβ induced an increase in the expression of COL1A1 and VIM, both in CAFs and in non-tumor NFs. Both THC and CBD ameliorated these effects. In summary, the results presented here reinforce the usefulness of cannabinoid agonists for the treatment of some relevant aspects of lung cancer pathology, and demonstrate in a novel way their possible effects on CAFs as a result of their relationship with cancer cells. Likewise, the results reinforce the usefulness of the combined use of THC and CBD, which has important advantages in relation to the possibility of using lower doses, thus minimizing the psychoactive effects of THC.

The cell–biomaterial interface is highly complex; thousands of molecules and many processes participate in its formation. Growing demand for improved biomaterials has highlighted the need to understand the structure and functions of this interface. Proteomic methods offer a viable alternative to the traditional in vitro techniques for analyzing such systems. Magnesium is a promoter of cell adhesion and osteogenesis. Here, we used the LC-MS/MS to compare the protein expression profiles of human osteoblasts (HOb) exposed to sol-gel coatings without (MT) and with Mg (MT1.5Mg) for 1, 3, and 7 days. PANTHER, DAVID, and IPA databases were employed for protein identification and data analysis. Confocal microscopy and gene expression analysis were used for further characterization. Exposure to MT1.5Mg increased the HOb cell area and the expression of SP7, RUNX2, IBP3, COL3A1, MXRA8, and FBN1 genes. Proteomic analysis showed that MT1.5Mg affected the early osteoblast maturation (PI3/AKT, mTOR, ERK/MAPK), insulin metabolism, cell adhesion (integrin, FAK, actin cytoskeleton regulation) and oxidative stress pathways. Thus, the effects of Mg on cell adhesion and osteogenesis are rather complex, affecting several pathways rather than single processes. Our analysis also confirms the potential of proteomics in biomaterial characterization, showing a good correlation with in vitro results. Funding for open access charge: CRUE-Universitat Jaume I This work was supported by Ministerio Ciencia e Innovación [PID2020-113092RB-C21]; Generalitat Valenciana [GRISOLIAP/2018/091, APOSTD/2020/036, PROMETEO/2020/069]; Universitat Jaume I [UJI-B2021-25]; and Basque Government [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, José Ortega, José Miguel Pedra and Iraide Escobés for their valuable technical assistance and Antonio Coso (GMI-Ilerimplant) for making the titanium discs.

This study investigated whether a 6-Watt ultraviolet C-lamp was capable of producing photofunctionalization on commercial implants during a medium observation term of 8 weeks. A total of 20 implants were inserted in 5 New Zealand rabbits, with each animal receiving 2 implants per tibia (one photofunctionalized and one untreated), according to a previously established randomization sequence. All implants were inserted by a single surgeon following the manufacturer's instructions. Histological analysis was performed by an evaluator who was blinded to the treatment condition. After 8 weeks of healing, the 2 groups showed no statistically significant differences in terms of bone-to-implant contact. Compared to control implants, the photofunctionalized implants showed improved wettability and more homogenous results. Within the limits of the present study, the use of this 6-W ultraviolet C-lamp, for an irradiation time of 15 minutes at a distance of 15 cm, did not improve the percentages of bone-to-implant contact in rabbits at an osseointegration time of 8 weeks.

The mechanisms of early failures in dental implant osseointegration are unclear. A possible cause of low levels of bone formation is lubricant contamination on implants during insertion. To explore the impact of lubricant contamination on dental implants, we used 5 New Zealand rabbits and inserted 2 implants per tibia in each animal for a total of 4 implants per animal (20 implants in total). In general, bicorticalization was achieved. The first implant was placed as suggested by the manufacturer with no lubricant used (control). The second implant was placed using a freshly lubricated contra-angle handpiece, which was used only for the test implants. Implant allocation was randomized, and the examining histologist was blinded to the results. All implants were placed by the same surgeon. The animals were maintained in accordance with animal experimentation guidelines. None of the implants failed to osseointegrate. Moreover, no significant difference was observed between the test and control groups. Based on the results of this study, the use of rotary instrument mineral oil lubricant did not jeopardize the osseointegration of dental implants in New Zealand rabbits.

Bone tissue provides support and protection to different organs and tissues. Aging and different diseases can cause a decrease in the rate of bone regeneration or incomplete healing; thus, tissue-engineered substitutes can be an acceptable alternative to traditional therapies. In the present work, we have developed an

The renal involvement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported. The etiology of kidney injury appears to be tubular, mainly due to the expression of angiotensin-converting enzyme 2, the key joint receptor for SARS-CoV-2; however, cases with glomerular implication have also been documented. The multifactorial origin of this renal involvement could include virus-mediated injury, cytokine storm, angiotensin II pathway activation, complement dysregulation, hyper-coagulation, and microangiopathy. We present the renal histological findings from a patient who developed acute kidney injury and de novo nephrotic syndrome, highly suggestive of acute IgA-dominant infection-associated glomerulonephritis (IgA-DIAGN) after SARS-CoV-2 infection, as evidenced by the presence of this virus detected in the renal tissue of the patient via immunohistochemistry assay. In summary, we document the first case of IgA-DIAGN associated to SARS-CoV-2. Thus, SARS-CoV-2 S may act as a super antigen driving the development of multisystem inflammatory syndrome as well as cytokine storm in patients affected by COVID-19, reaching the glomerulus and leading to the development of this novel IgA-DIAGN.

Cell culture of bone and tendon tissues requires mechanical stimulation of the cells in order to mimic their physiological state. In the present work, a device has been conceived and developed to generate a controlled magnetic field with a homogeneous gradient in the working space. The design requirement was to maximize the magnetic flux gradient, assuring a minimum magnetizing value in a 15 mm × 15 mm working area, which highly increases the normal operating range of this sort of devices. The objective is to use the machine for two types of biological tests: magnetic irradiation of biological samples and force generation on paramagnetic particles embedded in scaffolds for cell culture. The device has been manufactured and experimentally validated by evaluating the force exerted on magnetic particles in a viscous fluid. Apart from the magnetic validation, the device has been tested for irradiating biological samples. In this case, viability of human dental pulp stem cells has been studied in vitro after electromagnetic field exposition using the designed device. After three days of irradiation treatment, cellular microtissues showed a 59% increase in the viable cell number. Irradiated cells did not show morphological differences when compared with control cells.

To study the influence of the access window dimensions on the healing at the antrostomy and within the augmented maxillary sinus. A maxillary sinus augmentation was performed in twenty-four albino New Zealand rabbits. Antrostomies of 3 × 6 mm (small) or 5 × 6 mm (large) in dimensions were randomly prepared in each animal. A collagenated cortico-cancellous porcine bone was used to fill the elevated region, and an equine collagen membrane was placed on the antrostomies. Three different groups were formed, based on the time of euthanasia, i.e., 2, 4, and 8 weeks from surgery. No relevant changes of the height of the augmented sinus were detected over time. Mineralized bone increased between 2 and 4 weeks of healing while remained stable between 4 and 8 weeks. The highest amounts of new bone were found close to the sinus bone walls. No antrostomies were found healed with an even layer of corticalized bone, while large amounts of connective tissue were occupying the antrostomy in both groups. Antrostomies of different dimensions resulted in similar outcome in bone formation both in the antrostomy regions and within the elevated sinus.

Background/Objective Patients with non-small cell lung cancer (NSCLC) develop resistance to antitumor agents by mechanisms that involve the epithelial-to-mesenchymal transition (EMT). This necessitates the development of new complementary drugs, e.g., cannabinoid receptors (CB1 and CB2) agonists including tetrahydrocannabinol (THC) and cannabidiol (CBD). The combined use of THC and CBD confers greater benefits, as CBD enhances the effects of THC and reduces its psychotropic activity. We assessed the relationship between the expression levels of CB1 and CB2 to the clinical features of a cohort of patients with NSCLC, and the effect of THC and CBD (individually and in combination) on proliferation, EMT and migration in vitro in A549, H460 and H1792 lung cancer cell lines. Methods Expression levels of CB1, CB2, EGFR, CDH1, CDH2 and VIM were evaluated by quantitative reverse transcription-polymerase chain reaction. THC and CBD (10-100 mu M), individually or in combination (1:1 ratio), were used for in vitro assays. Cell proliferation was determined by BrdU incorporation assay. Morphological changes in the cells were visualized by phase-contrast and fluorescence microscopy. Migration was studied by scratch recolonization induced by 20 ng/ml epidermal growth factor (EGF). Results The tumor samples were classified according to the level of expression of CB1, CB2, or both. Patients with high expression levels of CB1, CB2, and CB1/CB2 showed increased survival reaching significance for CB1 and CB1/CB2 (p = 0.035 and 0.025, respectively). Both cannabinoid agonists inhibited the proliferation and expression of EGFR in lung cancer cells, and CBD potentiated the effect of THC. THC and CBD alone or in combination restored the epithelial phenotype, as evidenced by increased expression of CDH1 and reduced expression of CDH2 and VIM, as well as by fluorescence analysis of cellular cytoskeleton. Finally, both cannabinoids reduced the in vitro migration of the three lung cancer cells lines used. Conclusions The expression levels of CB1 and CB2 have a potential use as markers of survival in patients with NSCLC. THC and CBD inhibited the proliferation and expression of EGFR in the lung cancer cells studied. Finally, the THC/CBD combination restored the epithelial phenotype in vitro.

[EN] Purpose: To study the influence of scaffold properties on the organization of ¿in vivo¿ cartilage regeneration. Our hypothesis is that stress transmission to the cells seeded inside the scaffold pores or surrounding it, which is highly dependent on the scaffold properties, determine differentiation of both mesenchymal cells and dedifferentiated autologous chondrocytes. Methods: Four series of porous scaffolds made of different polyacrylate polymers, previously seeded with cultured rabbit chondrocytes or without cells preseeded, were implanted in cartilage defects in rabbits. Subchondral bone was always injured during the surgery in order to allow blood to reach the implantation site and fill scaffold pores. Results: Three months after implant, excellent tissue regeneration was obtained, with a well-organized layer of hyaline cartilage at the condylar surface in most cases of the hydrophobic or slightly hydrophilic series. The most hydrophilic material induced the poorest regeneration. However, few variations were observed between the preseeded and non-preseeded scaffolds. All the materials employed were biocompatible, biostable polymers, therefore, in contrast to other studies, our results are not perturbed by possible effects attributable to material degradation products, or to the loss of scaffold mechanical properties over time due to degradation. Conclusions: Cartilage regeneration mainly depends on the properties of the scaffold, such as stiffness and hydrophility, whereas little differences were observed between preseeded and non-preseeded scaffolds., Supported by the Ministry of Economy and Competitiveness through project No. MAT2013-46467-C4-R, including the FEDER financial support. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

The objective of this study was to test a regenerative medicine strategy for the regeneration of articular cartilage. This approach combines microfracture of the subchondral bone with the implant at the site of the cartilage defect of a supporting biomaterial in the form of microspheres aimed at creating an adequate biomechanical environment for the differentiation of the mesenchymal stem cells that migrate from the bone marrow. The possible inflammatory response to these biomaterials was previously studied by means of the culture of RAW264.7 macrophages. The microspheres were implanted in a 3 mm-diameter defect in the trochlea of the femoral condyle of New Zealand rabbits, covering them with a poly(l-lactic acid) (PLLA) membrane manufactured by electrospinning. Experimental groups included a group where exclusively PLLA microspheres were implanted, another group where a mixture of 50/50 microspheres of PLLA (hydrophobic and rigid) and others of chitosan (a hydrogel) were used, and a third group used as a control where no material was used and only the membrane was covering the defect. The histological characteristics of the regenerated tissue have been evaluated 3 months after the operation. We found that during the regeneration process the microspheres, and the membrane covering them, are displaced by the neoformed tissue in the regeneration space toward the subchondral bone region, leaving room for the formation of a tissue with the characteristics of hyaline cartilage., Facultad de Ciencias Exactas, Laboratorio de Investigación en Osteopatías y Metabolismo Mineral

Background: Due to the lack of a gold standard diagnostic test, reference centres with experienced personnel and costly procedures are needed for primary ciliary dyskinesia (PCD) diagnostics. Diagnostic flowcharts always start with clinical symptoms. Therefore, the aim of this work is to define differential clinical criteria so that only patients clinically compatible with PCD are referred to reference centres. Materials and methods: 18 variables from 476 Mediterranean patients with clinically suspicious PCD were collected. After analysing cilia function and ultrastructure, 89 individuals were diagnosed with PCD and 387 had a negative diagnosis. Simple logistic regression analysis, considering PCD as a dependent variable and the others as independent variables, was done. In order to define the variables that best explain PCD, a step-wise logistic regression model was defined. Aiming to classify individuals as PCD or PCD-like patients, based on variables included in the study, a classification and regression tree (CART) was designed. Results and conclusions: Simple logistic regression analysis shows statistically significant association between age at the beginning of their symptomatology, periodicity, fertility, situs inversus, recurrent otitis, atelectasis, bronchiectasis, chronic productive cough, rhinorrea, rhinusinusitis and recurrent pneumonias, and PCD. The step-wise logistic regression model selected situs inversus, atelectasis, rhinorrea, chronic productive cough, bronchiectasis, recurrent pneumonias, and otitis as PCD predictive variables (82% sensitivity, 88% specificity, and 0.92 Area Under the Curve (AUC)). A decision tree was designed in order to classify new individuals based on pansinusitis, situs inversus, periodicity, rhinorrea, bronchiectasis, and chronic wet cough.

Background The aim of this study was to conduct histological analysis of a human tooth resected with the periodontal insertion apparatus intact following treatment using biologically oriented preparation technique (BOPT). Material and methods This descriptive histological dento-periodontal study used an anterior tooth extracted with the surrounding periodontal tissues intact, following prosthetic restoration with BOPT. The sample patient was recruited from among those attending the Department of Dental Medicine at the Faculty of Medicine and Dentistry, University of Valencia (Spain). Eight serial sections of the restored tooth were processed. The relative location and histological characteristics of the cemented prosthetic crown, the dental tissues of the tooth prepared by BOPT technique, and the periodontal tissues were analyzed. Results Structural analysis of the neoformed junctional epithelium showed that the number of layers decrease apically until there was a single row of cells perfectly adhered to the acellular cementum, and beneath the epithelium a connective tissue evidently free from inflammation. The tissues of the neoformed periodontium (gingival ligament, sulcular epithelium, junctional epithelium) presented histologic normality. Conclusions Biologically oriented preparation technique is a reliable alternative to conventional horizontal finish lines. Key words:Vertical preparation, prosthetic cementoenamel junction (PCEJ), finish line, BOPT, crown.

Dental implantology is still an expanding field of scientific study because of the number of people that receive dental therapies throughout their lives worldwide. Recovery times associated to dental surgery are still long and demand strategies to improve integration of metallic devices with hard tissues. In this work, an in vitro ceramic coating is proposed to improve and accelerate osseointegration of titanium surfaces conceived to be used as dental implants or hip or knee prosthesis, shaped either as dishes or screws. Such coating consists of hydroxyapatite microdomains on the implant surfaces obtained in vitro by immersion of titanium alloy samples (Ti6Al4V) in a simulated body fluid. This titanium alloy is highly used in implant dentistry and trauma surgery, among other fields. Once the immersion times under physiological conditions yielding to different ceramic topographies on this alloy were set, the acellular coating time of major interest so as to optimize its biological development was determined. For this purpose, dental pulp mesenchymal cells were cultured on titanium coated surfaces with different hydroxyapatite outline, and cell adhesion, proliferation and morphology were followed through histological techniques and scanning electron microscopy. It was found that 4 days of acellular hydroxyapatite coating led to a significant cell adhesion on the titanium alloys at an early stage (6 h). Cells tended although to detach from the surface of the coating over time, but those adhered on domains of intricated topography or hydroxyapatite cauliflowers proliferated on them, leading to isolated large cell clusters. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2723-2729, 2016.

Articular cartilage in synovial joints is a hyaline cartilage highly hydrated with a rigorous order of cells and fibers and a specific content of proteoglycans and glycoproteins. It provides a low-friction surface, participates in the lubrication of the synovial joints, and distributes the forces to the underlying bone. It is an avascular and aneural tissue where small metabolites diffuse to and from cells. Unlike hyaline cartilage in other locations, articular cartilage lacks perichondrium, a layer of fibrous tissue around it that serves as the source of new cartilage cells. Thus, although extracellular matrix undergoes continuous remodeling throughout life, the ability to repair is limited, and often, degeneration in weight-bearing joints occurs by age or due to injuries, causing a reduction in mobility and increasing pain with joint movement, in a clinical profile called osteoarthritis, eventually progressing to long-term disability. Several medical and orthopedic treatments have been developed, but their long-term results are not usually satisfactory. New approaches point to tissue engineering techniques, where the combination of scaffolds made of biomaterials as artificial extracellular matrix, along with autologous or allogeneic cells and growth factors are used. Multiple scaffolds have been tested for articular cartilage regeneration, with different composition of biomaterial, conformations, and mechanical properties. With respect to the cellular source, autologous chondrocytes have been used, after an in vitro expansion, as well as mesenchymal stem cells from different origins (such as synovial membrane, bone marrow or adipose tissue), along with factors that induce the growth and cellular differentiation toward chondrogenic cells. We present our research in this field and summarize the advances obtained in the regeneration of articular cartilage.

Dental implants can be integrated at both hard and soft tissue levels. Osseointegration is a dynamic process during its establishment and maintenance, characterized by resorption and apposition events, and the extent and degree of osseointegration is in part affected by implant surface configuration. It is marked by three distinct healing stages during endosseous implant integration: osteoconduction, “de novo” bone formation, and bone remodeling, which occur by the interaction of different bone cells: osteocytes, osteoblasts, and osteoclasts. One of the most important aspects for reaching clinical osseointegration is the primary stability during implant placement. The extent of primary anchorage is tightly related to native bone characteristics, implant design, patient characteristics, and surgical technique, respectively, where they all regulate to some extent the strain applied to mineralized tissue and directly related to bone interfacial stress and frictional force transferred, which is clinically interpreted as insertion torque. At 2 weeks there is an evident accelerated proliferation of blood vessels and mesenchymal cells embedded inside the provisional granulation tissue that stimulate the formation of an immature peri-implant primary bone or woven bone. At 4 weeks, the woven bone replacement by lamellar bone suggests an initial remodeling has begun, and it is evidenced by the presence of primary osteonic structures that elucidate the onset of woven bone remodeling toward lamellar bone configuration surrounding blood vessels. At 8 weeks, the healing chambers clearly show a “bone remodeling” process, with the presence of parallel fibered lamellar bone deposition, and there is plenty of bone formed, evidenced by the presence of primary and secondary osteonic structures. Lamellar osteons (haversian systems) outlined by cement-line boundaries are visible.

Odontoblasts are post-mitotic cells responsible for maintenance of the dentin, and are therefore important for dental health. In some cases, irreversible pulpitis leads to necrosis and consequently death of odontoblasts. Regenerative endodontics (RE) uses the concept of tissue engineering to restore the root canals to a healthy state, allowing for continued development of the root and surrounding tissue. Human dental pulp stem cells (hDPSCs) have been successfully used in RE to restore odontoblast function. Surface microgeometry is one of the most important factors involved in the induction of differentiation of hDPSCs into odontoblast-like cells. Although different authors have demonstrated the importance of a dentin-like surface with accessible dentin tubules to induce differentiation of hDPSCs, the ultrastructural characteristics of the cells and the secreted extracellular matrix have not been studied in depth. Here, we used an acellular dentin scaffold containing dentin tubules in different spatial geometries, which regulated their accessibility to cells. hDPSCs were cultured on the scaffolds for up to 6 weeks. Systematic characterization of differentiated cells was performed using both optical (hematoxylin and eosin, Masson trichrome, and immunohistochemical determination of dentin sialoprotein [DSSP]) and transmission electron microscopy. The results presented here indicated that cells grown on the dentin surface containing accessible dentin tubules developed a characteristic odontoblastic phenotype, with cellular processes similar to native odontoblasts. The cell organization and characteristics of secreted extracellular matrix were also similar to those of native dentin tissue. Cells grown on non-accessible dentin tubule surfaces secreted a more abundant and dense extracellular matrix, and developed a different phenotype consisting of secretory flat cells organized in layers. Cells grown far from the scaffold, i.e., directly on the culture well surface, developed a secretory phenotype probably influenced by biochemical factors released by the dentin scaffold or differentiated cells. The results presented here support the use of hDPSCs to regenerate dentin and show the utility of scaffold microgeometry for determining the differentiation and secretory phenotype of cultured cells.

There is an ever-increasing need to develop dental implants with ideal characteristics to achieve specific and desired biological response in the scope of improve the healing process post-implantation. Following that premise, enhancing and optimizing titanium implants through superficial treatments, like silica sol-gel hybrid coatings, are regarded as a route of future research in this area. These coatings change the physicochemical properties of the implant, ultimately affecting its biological characteristics. Sandblasted acid-etched titanium (SAE-Ti) and a silica hybrid sol-gel coating (35M35G30T) applied onto the Ti substrate were examined. The results of in vitro and in vivo tests and the analysis of the protein layer adsorbed to each surface were compared and discussed. In vitro analysis with MC3T3-E1 osteoblastic cells, showed that the sol-gel coating raised the osteogenic activity potential of the implants (the expression of osteogenic markers, the alkaline phosphatase (ALP) and IL-6 mRNAs, increased). In the in vivo experiments using as model rabbit tibiae, both types of surfaces promoted osseointegration. However, the coated implants demonstrated a clear increase in the inflammatory activity in comparison with SAE-Ti. Mass spectrometry (LC–MS/MS) analysis showed differences in the composition of protein layers formed on the two tested surfaces. Large quantities of apolipoproteins were found attached predominantly to SAE-Ti. The 35M35G30T coating adsorbed a significant quantity of complement proteins, which might be related to the material intrinsic bioactivity, following an associated, natural and controlled immune response. The correlation between the proteomic data and the in vitro and in vivo outcomes is discussed on this experimental work.

Osteoarthritis is an inflammatory disease in which all joint-related elements, articular cartilage in particular, are affected. The poor regeneration capacity of this tissue together with the lack of pharmacological treatment has led to the development of regenerative medicine methodologies including microfracture and autologous chondrocyte implantation (ACI). The effectiveness of ACI has been shown in vitro and in vivo, but the use of other cell types, including bone marrow and adipose-derived mesenchymal stem cells, is necessary because of the poor proliferation rate of isolated articular chondrocytes. In this investigation, we assessed the chondrogenic ability of human dental pulp stem cells (hDPSCs) to regenerate cartilage in vitro and in vivo. hDPSCs and primary isolated rabbit chondrocytes were cultured in chondrogenic culture medium and found to express collagen II and aggrecan. Both cell types were cultured in 3% alginate hydrogels and implanted in a rabbit model of cartilage damage. Three months after surgery, significant cartilage regeneration was observed, particularly in the animals implanted with hDPSCs. Although the results presented here are preliminary, they suggest that hDPSCs may be useful for regeneration of articular cartilage.

Introduction: Primary ciliary dyskinesia (PCD) is a rare genetic disease with an estimated prevalence of 1:20.000 births. It is characterized by abnormal motility of cilia, leading to impaired mucociliary clearance, and subsequent infection and chronic inflammation of the airways. PCD also affects spermatozoa and cilia in the Fallopian tubes, contributing to fertility issues; dyskinesia of embryonic nodal cilia causes a random distribution of the organs. Areas covered: An overview of the history, genetics, clinical manifestations in children and adults, diagnostic tests, treatments, and prognosis are reviewed. We also discuss current research and future prospects of PCD. Expert opinion: As PCD comprises defects in all organs with motile cilia, patients have a variety of clinical manifestations, often characterized by their presence from birth. Because of the non-specific symptoms, PCD is often confused with other diseases such as cystic fibrosis. There is no gold standard diagnostic test and a variety of diagnostic tests are required, including high-speed video analysis and transmission electron microscopy. Reanalysis following primary cultures of the epithelial cells can help to differentiate primary from secondary defects. Despite being a genetic disease, due to the genetic heterogeneity of PCD, gene analysis can currently only explain 65% of the cases. There is no treatment for PCD, and therapeutic options that contribute to the wellbeing of the patients are based on expert opinion.

Spanish Ministry of Health Spanish Ministry of Science and Innovation Fundacio Marato de TV3, Spain

Perinatal stem cells such as human umbilical cord Wharton's jelly stem cells (HWJSCs) are excellent candidates for tissue engineering because of their proliferation and differentiation capabilities. However, their differentiation potential into epithelial cells at in vitro and in vivo levels has not yet been reported. In this work we have studied the capability of HWJSCs to differentiate in vitro and in vivo to oral mucosa and skin epithelial cells using a bioactive three-dimensional model that mimics the native epithelial-mesenchymal interaction. To achieve this, primary cell cultures of HWJSCs, oral mucosa, and skin fibroblasts were obtained in order to generate a three-dimensional heterotypical model of artificial oral mucosa and skin based on fibrin-agarose biomaterials. Our results showed that the cells were unable to fully differentiate to epithelial cells in vitro. Nevertheless, in vivo grafting of the bioactive three-dimensional models demonstrated that HWJSCs were able to stratify and to express typical markers of epithelial differentiation, such as cytokeratins 1, 4, 8, and 13, plakoglobin, filaggrin, and involucrin, showing specific surface patterns. Electron microscopy analysis confirmed the presence of epithelial cell-like layers and well-formed cell-cell junctions. These results suggest that HWJSCs have the potential to differentiate to oral mucosa and skin epithelial cells in vivo and could be an appropriate novel cell source for the development of human oral mucosa and skin in tissue engineering protocols.

Background/Objective: Tracheal stenosis produces serious dyspnea. In some cases, a defective correction or timely intervention can lead to the death of the patient. The Hospital Clinico Universitario de Valencia is a reference hospital for the treatment of patients with airway problems exhibiting complex tracheal stenosis. Surgery is limited to a maximum length of 6-7 cm and it is not exempt of mortality (3-5%). In this study we have study the useful of decellularized porcine tracheas to be used in airway regenerative medicine. Methods: Three porcine tracheas were cut in 3 cm rings and exposed to SDS 2% for up to four weeks. Cell content was evaluated by fluorescence microscopy (DAPI) weekly. Haematoxylin Eosin, Masson Trichrome, Orcein and PAS staining. Tracheal rings were cultured with human airway epithelial cells and chondrocytes for up to three weeks. Results : SDS removed completely cells from tracheas analysed. After culture we observed epithelial cell attachment in the surface of the tracheal rings as well as chondrocytes in the cartilage layer of the tracheas analysed. Conclusion : Our results point to the utility of SDS 2% treatment to remove the cells in the different tissues forming the trachea. This treatment not only exhibits a minimal disturbance of the extracellular matrix but also allows the attachment and proliferation of airway epithelial cells and primary chondrocytes. In summary, SDS may be useful to generate decellularized scaffolds for airway tissue engineering applications.

Objective To investigate the effect of antiangiogenic treatment on experimental endometriotic lesion nerve fibers. Design Heterologous mouse model of endometriosis. Setting University Institute IVI, University Hospital La Fe. Animal(s) Ovariectomized nude mice (n = 16) receiving human endometrial fragments from oocyte donors (n = 4). Intervention(s) Endometrium fragments stuck in the peritoneum of 5-week-old female nude mice treated with vehicle (n = 8) and antiangiogenic agent cabergoline (n = 8; Cb 2, 0.05 mg/kg/day) for 14 days. Main Outcome Measure(s) Immunofluorescence analysis of von-Willebrand factor (vWF) and vascular smooth muscle cells (αSMA) for evaluating the number of immature blood vessels (IBV) and microvascular density (MVD); immunochemical analysis of protein-gene product 9.5 (PGP 9.5) to assess nerve fibers density (NFD), and blue toluidine staining to confirm presence of mast cells and macrophages in endometriotic lesions. Result(s) All the results were quantified by morphometric techniques. The IBV, NFD, and number of macrophages and mast cells were statistically significantly decreased in the Cb2-treated group when compared with controls. Conclusion(s) Antiangiogenic treatment statistically significantly diminishes new blood vessel formation after macrophage, mast cell, and nerve fiber reduction, providing a rationale to test antiangiogenic agents as a novel therapeutic approach to severe pelvic pain associated with human peritoneal endometriosis.

Resumen Introduccion La discinesia ciliar primaria (DCP) es una entidad clinica de dificil diagnostico debido a que la motilidad y la estructura ciliar pueden variar segun los pacientes. El objetivo principal ha sido evaluar la sensibilidad y especificidad para el diagnostico de la DCP de un sistema de videoanalisis de alta resolucion digital junto con un software propio desarrollado por nosotros para el analisis de la movilidad ciliar (Desinsoft-Bio 200). El objetivo secundario ha sido relacionar la actividad nasal ciliar con la clinica y las anomalias estructurales del cilio respiratorio. Material y metodos Se analizaron el transporte mucociliar mediante un metodo isotopico, la ultraestructura con microscopia electronica y la frecuencia y el patron de batida ciliar mediante el sistema Desinsoft-Bio 200. Veinticinco pacientes con DCP (11 de ellos con sindrome de Kartagener), 27 con discinesia ciliar secundaria y 34 sanos. Resultados El transporte mucociliar fue defectuoso en los pacientes con DCP y discinesia ciliar secundaria. La inmovilidad ciliar se observo en 6 de los pacientes con sindrome de Kartagener correlacionandose con la ausencia de dineina. Un movimiento ciliar alterado (discinesia) acontecio en el resto de pacientes con DCP, correlacionandose con un deficit parcial de dineina o ulraestructura normal. La movilidad ciliar y la estructura fueron normales en las discinesias ciliares secundarias y en sanos. Discusion y conclusiones El transporte mucociliar nasal posee una gran sensibilidad cercana al 100% para el diagnostico de la DCP, pero muy baja especificidad. El sistema de analisis de la movilidad ciliar mediante el sistema de video digital de alta velocidad y resolucion tiene una sensibilidad y especificidad elevadas para el diagnostico de la DCP. Este sistema de videoanalisis es mas util que el estudio ultraestructural y el transporte mucociliar como cribado de la DCP. La ausencia de dineina se correlaciona con la inmovilidad ciliar y es mas frecuente en pacientes con sindrome de Kartagener.

Previous studies in an experimental mouse model of endometriosis have shown that the dopamine agonist (DA) cabergoline (Cb2) reduces angiogenesis and endometriotic lesions, hypothetically binding to the dopamine receptor type-2 (DRD2). To date, this has not been described in human endometrium and/or endometriotic lesions. Thus, we aimed to investigate the presence of DRD2 in said tissues. Endometrium fragments were implanted in nude mice treated with different doses of Cb2. Polymerase chain reaction assays and immunohistochemistry were performed to analyze the gene and protein expressions (respectively) of DRD2, VEGF, and VEGF receptor-2 (KDR). In addition, lesions and endometrium from women with mild and severe endometriosis and endometrium from healthy women were collected to analyze their gene expression profile. In experimental endometriosis, DRD2 was expressed at gene and protein levels in all three groups. VEGF gene and protein expressions were significantly lower in lesions treated with Cb2 than in controls. KDR protein expression was significantly lower in experimental lesions treated with Cb2 than in controls. In eutopic endometria, there was a significant decrease in DRD2 expression and an increase in VEGF in women with mild and severe endometriosis with respect to healthy patients. In endometriosis, KDR expression was significantly higher in red than in white and black lesions. VEGF expression was significantly lower in black than in red lesions. DRD2 is present in the human eutopic and ectopic endometrium and is regulated by DA, which provides the rationale for pilot studies to explore its use in the treatment of endometriosis.

J Oral Pathol Med (2010) 39: 10–15 Background: Among the agents that cause parotid sialosis, diabetes mellitus type 2 and chronic alcoholism are included. In this study, the morphometrical modifications in the diabetic parotid sialosis were determined to compare them with the histopathological characteristics of alcoholic parotid sialosis. Methods: Five parotid biopsy samples obtained from patients with diabetic sialosis, 12 samples from patients with alcoholic sialosis and seven from individuals without these pathologies (control group) were analyzed. A morphometrical study of parotid parenchyme and stroma, using a digital image analyzer attached to an optical microscope, was carried out. Dimensions of serous acini and striated ducts, the area occupied by the fatty tissue, and the number of ducts were recorded. Mean values were compared using the Mann–Whitney U-test (P ≤ 0.05). Results: The variables analyzed in diabetic patients did not show significant differences with respect to the control group. However, when diabetics were compared with alcoholics, the alcoholics exhibited a noticeable reduction in the proportion of fatty tissue of stroma and a significant development of ductal epithelium that contributed to increase the caliber of the striated ducts. Conclusions: These results indicate that the glandular hypertrophy in the diabetic parotid sialosis is not directly associated with the ductal and acinar size, amount of fatty tissue and ductal hyperplasy. Nevertheless, these findings show that the ductal dimensions and the proportion of adipose tissue are variables that allow us to establish histopathological differences between diabetic and alcoholic sialosis.

Survival of newly implanted retrograde-shed endometrial tissue during menstruation in an ectopic location requires an adequate blood supply. This suggests that angiogenesis is a prerequisite for the development of endometriosis and that its inhibition may be a target for preventing development. Previous studies have shown that vascular endothelial growth factor (VEGF), a heparin-binding glycoprotein, has an essential role in angiogenesis. The main regulatory factor for angiogenesis appears to be binding of VEGF to its type-2 receptor (VEGFR-2). Cabergoline (Cb2) and other dopamine agonists promote endocytosis of the VEGF receptor-2 (VEGFR-2) in endothelial cells, thereby preventing VEGF-VEGFR-2 binding and reducing neoangiogenesis. This study used an experimental model of endometriosis to assess the anti-angiogenic effects of Cb2 at the morphological level on growth of established endometriotic lesions and to investigate molecular mechanisms associated with these effects. The investigators implanted human endometrial sections in the peritoneum of nude mice and divided the mice into 3 groups which received by gavage either Cb2 in daily oral doses of 0.05 (low-dose group) or 0.1 (high-dose group) mg/kg, or a control vehicle for 14 days. After treatment, the implants were processed to compare proliferative activity, the neoangiogenic process, angiogenic gene expression, and VEGFR-2 phosphorylation in the 3 groups. Compared with the controls, there was a significant decrease in the percentage of active endometriotic lesions in both Cb2-treated groups (P < 0.05). The cellular proliferation index was also decreased (P < 0.001). Histology showed intact lesions in the controls, whereas the Cb2-treated lesions had a lack of cellularity and organization characteristic of atrophic or degenerative tissue. In addition, Cb2 treatment impaired the neoangiogenic process at the morphological level as shown by reduced formation of new blood vessels in endometriotic lesions and at the molecular level by inhibition of VEGF expression. Compared with the controls, there was a significant reduction in the degree of total VEGFR-2 phosphorylation (P < 0.001) in mice treated with Cb2. The investigators believe that these findings, together with previous evidence, support targeting of the angiogenic process through blockade of VEGFR-2 as a potentially successful approach to treatment of peritoneal endometriosis in humans.

Liquid nitrogen is the most common medium used by tissue banks for the storage of cryopreserved heart valves. This study evaluates the effect of the length of storage on human cryopreserved heart valves. Human tissues (14 aortic and 13 pulmonary) were frozen in a controlled-rate freezer (1 degrees C/min) and stored in the liquid phase of a nitrogen tank for 9.1+/-1.6 years. The preservative solution was medium M199 containing 5% human serum albumin and 10% Me(2)SO. After thawing in a water bath at 42 degrees C, the cryoprotectant was removed. Then, fragments from vascular wall and leaflet were dissected. Explant cultures and histological studies were performed in order to assess cell viability and structural integrity. CD90 and CD31 expression was analysed in cultured cells using flow cytometry. Light microscopy, immunofluorescence staining and laser scanning confocal microscopy were used to evaluate cell viability and extracellular matrix components. Electron microscopy was used for ultrastructural study. Cell cultures could be obtained from all the specimens assayed. Cells grew from explants showing a fibroblastic phenotype. CD90 expression was common in cultured cells but a low percentage of cells expressed CD31. Histological results showed a good preservation estructure in both leaflets and vascular walls. Morphological features of cellular irreversible damage were very rare. No differences which could be due to length of allograft storage period were observed. We concluded that allografts stored in liquid nitrogen up to 13 years did not significantly undergo loss of cell viability other than that due to disinfection, freezing and thawing protocols.

Introduccion El estudio histologico de los polipos nasals no muestra lesiones especificas, pero la infiltracion de la mucosa por eosinofilos parece ser una caracteristica de la poliposis nasal. El objetivo del trabajo es estudiar las posibles relaciones entre algunos aspectos histologicos y clinicos en la poliposis nasal. Asimismo pretendemos comparar la cuantificacion de la eosinofilia tisular segun el numero de eosinofilos por campo con la obtenida porcentualmente respecto al total de celulas inflamatorias. Material y metodo Hemos estudiado a 40 pacientes con poliposis nasal bilateral idiopatica y valoramos las correlaciones existentes entre diversos aspectos clinicos, como el estadio endoscopico y el radiologico, la asociacion con asma e intolerancia a antiinflamatorios no esteroideos, con algunos aspectos histologicos de los polipos nasales, como la frecuencia de metaplasia, fibrosis y grado de infiltracion eosinofilica. Disponemos de un grupo control de 12 sujetos con mucosa nasal sana para la comparacion de nuestros resultados. Resultados La eosinofilia tisular se correlaciona estadisticamente con el estadio clinico y presenta una tendencia a ser mas alta en el sindrome de ASA triada. La medida de la eosinofilia tisular de forma cuantitativa (numero de eosinofilos por campo) se correlaciona con la obtenida porcentualmente (respecto al total de celulas inflamatorias del infiltrado). Conclusiones La infiltracion por eosinofilos de la mucosa nasal es, junto con el edema, la caracteristica histological mas constante de la poliposis nasal y parece ser un factor importante en el comportamiento clinico de la poliposis nasosinusal. La medicion cuantitativa de la eosinofilia tisular es mas rapida y sencilla y con la misma significacion que la porcentual.

Background: In alcoholic parotid sialosis, the gland is frequently enlarged due to ductal and/or acinar hypertrophy, ductal hyperplasy and stromal fat infiltration. The aim of this study was to determine acinar and ductal dimensions, the number of striated ducts and the proportion of fat tissue in patients with and without alcoholic parotid sialosis. Methods: Twelve parotid biopsy samples from patients with hepatic alcoholic cirrhosis and those from seven controls were used. A morphometrical study with a digital image analyser attached to an optical microscope was carried out. Direct and indirect indicators from acinar and ductal dimensions were recorded. The number of striated ducts and the proportion of fat tissue in stroma were determined. Fifteen records for each variable were taken. Mean values were compared using the Mann–Whitney U-test (P ≤ 0.05). Results: There were no significant differences in acinar and ductal dimensions and the number of striated ducts between alcoholic and control patients. The proportion of fat tissue in alcoholic parotides was significantly lower than that in the controls. Conclusions: These results do not corroborate previous qualitative descriptions about acinar and ductal hypertrophy and ductal hyperplasy in alcoholic patients. The main cause of parotid enlargement could not be stromal fat infiltration. The data could be used for differential diagnoses of sialosis.

Introduction The histological study of nasal polyps does not reveal any specific lesions but eosinophilic infiltration nasal mucosae seems to be characteristic of nasal polyposis. The aim of this work is to study possible links between certain histological and clinical aspects in nasal polyposis. Furthermore, we attempt to compare the quantification of tissue eosinophilia according to the number of eosinophils per field with the percentage figure obtained with respect to the total of inflammatory cells. Material and method We have studied 40 patients with idiopathic bilateral nasal polyposis, assessing the correlations between various clinical aspects such as their endoscopic and radiological status, association with asthma and intolerance to NSAIDs, against histological aspects of nasal polyps such as the frequency of metaplasia, fibrosis, and the degree of eosinophilic infiltration. A group of 12 healthy subjects allowed comparison of our results with healthy nasal mucosa. Results Tissue eosinophilia correlates statistically with clinical staging and tends to be higher in patients with ASA triad. The quantitative measurement of tissue eosinophilia (number of eosinophils per field) correlates with the percentage figure obtained (with respect to the total number of inflammatory cells in the infiltrate). Conclusions Eosinophil infiltration of the nasal mucosa is, together with oedema, the most constant histological characteristic of nasal polyposis and seems to be an important factor in the clinical behaviour of sinonasal polyposis. Quantitative measurement of tissue eosinophilia is easier and quicker to perform and equivalent to percentage evaluation.