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A new in vitro human corneal epithelial model (QobuR) obtained from normal limbal tissue has been developed to study ocular irritancy of different ophthalmic compounded drugs. Phenotypical characterization and trans-epithelial electrical resistance (TEER) of QobuR revealed essential similarities compared with a native human cornea, displaying functional markers and TEER values near 1500 Ωcm2 at day 7th of cellular differentiation. Using this model, ocular irritancy and barrier integrity alterations were evaluated using MTT reaction and variations in TEER. We found that some of the Non-Irritant products evaluated still damage the corneal epithelial integrity and current protocols for ocular irritancy should therefore include a barrier integrity evaluation. Moreover, in order to comprehensively evaluate corneal permeability of the active ingredients, we propose the use of QobuR as an all-in-one alternative method for evaluating ocular irritancy, barrier disruptions and permeability rates of topically applied ocular drugs to improve current in vitro drug testing procedures.

Tissue engineering is one of the fields of clinical medicine that has forged ahead in recent years, especially because of its role as a potential alternative to organ transplantation. The main aim of this study has been the development of biocompatible materials to form extracellular matrix (ECM) structures in order to provide the necessary conditions for the settlement, proliferation and differentiation of dermal cells such as fibroblasts. To this end, human plasma gels were synthesized with the addition of increasing concentrations of transglutaminase (TGase), which catalyses the formation of covalent bonds between Lys and Glu residues. These materials were structurally characterized using rheology and texturometry and were found to have good structural resistance and elasticity for fibroblast culture. A remarkable improvement in the mechanical properties of the human plasma gels was detected when the two highest TGase concentrations were tested, which may be interpreted as an increase in the number of covalent and non-covalent bonds formed between the plasma protein chains. Furthermore, a human fibroblast primary culture was seeded on human plasma scaffolds and satisfactorily proliferated at 37 °C. This was verified in the images obtained by optical microscopy (OM) and by scanning electron microscopy (SEM), which confirmed that the structure of this type of material is suitable for the growth and proliferation of dermal fibroblasts.

Biological membranes are currently used in Ophthalmology in order to treat different ocular disorders. These membranes have different properties such as cellular biocompatibility and promoting wound healing. Moreover, intrinsic antimicrobial properties could also be desirable because it would allow their use reducing the risk of infections. Graphene and its derivatives are promising biomaterials that already proved their bactericidal effect. However, their clinical use is limited due to the controversial results regarding their toxicity. In this work, we have developed and characterized a reduced graphene oxide membrane (rGOM) for its use in ocular Regenerative Medicine, and studied its in vitro and in vivo biocompatibility and genotoxicity with different types of human ocular cells. We proved that rGOM allowed the growth of different ocular cells without inducing in vitro or in vivo cytotoxicity or genotoxicity in the short-term. These results indicate that rGOM may be a promising candidate in Regenerative Medicine for the treatment of different ocular pathologies., This work was supported by Consejería de Economía y Empleo del Principado de Asturias: [grant number: IDE/2014/000737 and IDE/2015/000889].

Letter to the Editor.-- et al.

Although the expression of the chemokine receptor CCR1 has been demonstrated in several structures related to nociception, supporting the nociceptive role of chemokines able to activate it, the involvement of CCR1 in neoplastic pain has not been previously assessed. We have assayed the effects of a CCR1 antagonist, J113863, in two murine models of neoplastic hyperalgesia based on the intratibial injection of either NCTC 2472 fibrosarcoma cells, able to induce osteolytic bone injury, or B16-F10 melanoma cells, associated to mixed osteolytic/osteoblastic bone pathological features. The systemic administration of J113863 inhibited thermal and mechanical hyperalgesia but not mechanical allodynia in mice inoculated with NCTC 2472 cells. Moreover, in these mice, thermal hyperalgesia was counteracted following the peritumoral (10-30μg) but not spinal (3-5μg) administration of J113863. In contrast, hyperalgesia and allodynia measured in mice inoculated with B16-F10 cells remained unaffected after the administration of J113863. The inoculation of tumoral cells did not modify the levels of CCL3 at tumor or spinal cord. In contrast, although the concentration of CCL5 remained unmodified in mice inoculated with B16-F10 cells, increased levels of this chemokine were measured in tumor-bearing limbs, but not the spinal cord, of mice inoculated with NCTC 2472 cells. Increased levels of CCL5 were also found following the incubation of NCTC 2472, but not B16-F10, cells in the corresponding culture medium. The intraplantar injection of CCL5 (0.5ng) to naïve mice evoked thermal hyperalgesia prevented by the coadministration of J113863 or the CCR5 antagonist, d-Ala-peptide T-amide (DAPTA), demonstrating that CCL5 can induce thermal hyperalgesia in mice through the activation of CCR1 or CCR5. However, contrasting with the inhibitory effect evoked by J113863, the systemic administration of DAPTA did not prevent tumoral hyperalgesia. Finally, the peritumoral administration of an anti-CCL5 antibody completely inhibited thermal hyperalgesia evoked by the inoculation of NCTC 2472 cells.

Objective We report clinical and functional outcomes obtained after application of an autologous bioengineered composite skin (ABCS) produced in a single Spanish tissue-engineering unit. Materials/methods Twenty-five burned patients treated with ABCS from 1999 to 2007 in five burn centres were included in the study. Mean age was 29 years (SD 11), with mean total body surface area (TBSA) burned being 74% (SD 17) and mean full-thickness injury of 61% (SD 19) of TBSA. Results The mean area initially engrafted with ABCS was 24% (SD 13) of TBSA, with a final take of 49% (SD 30, range 0–100%). ABCS achieved permanent coverage of a mean of 11% (SD 8) of TBSA. In subset analyses, lack of pre- and post-application wound bed infection and lack of serious acute systemic complications at the time of engraftment were significantly associated with better ABCS take. Conclusions Final take obtained with ABCS could be improved with the use of non-cytotoxic topical antibiotics following engraftment. The use of plasma to prepare ABCS reduces production costs: cost-effectiveness ratio is not a limitation for its use. In terms of patient satisfaction, cosmetic/functional outcomes (general appearance, texture, flexibility, sensitivity and colour) of ABCS and split-thickness autografts are not different statistically.

Over the past few years, whole skin xenotransplantation models that mimic different aspects of psoriasis have become available. However, these models are strongly constrained by the lack of skin donor availability and homogeneity. We present in this study a bioengineering-based skin-humanized mouse model for psoriasis, either in an autologous version using samples derived from psoriatic patients or, more importantly, in an allogeneic context, starting from skin biopsies and blood samples from unrelated healthy donors. After engraftment, the regenerated human skin presents the typical architecture of normal human skin but, in both cases, immunological reconstitution through intradermal injection in the regenerated skin using in vitro-differentiated T1 subpopulations as well as recombinant IL-17 and IL-22 Th17 cytokines, together with removal of the stratum corneum barrier by a mild abrasive treatment, leads to the rapid conversion of the skin into a bona fide psoriatic phenotype. Major hallmarks of psoriasis were confirmed by the evaluation of specific epidermal differentiation and proliferation markers as well as the mesenchymal milieu, including angiogenesis and infiltrate. Our bioengineered skin-based system represents a robust platform to reliably assess the molecular and cellular mechanisms underlying the complex interdependence between epidermal cells and the immune system. The system may also prove suitable to assess preclinical studies that test the efficacy of novel therapeutic treatments and to predict individual patient response to therapy.

Using a recently described skin-humanized model based on the engraftment of human bioengineered skin equivalents onto immunodeficient mice, we compared the efficacy of different in vivo gene transfer strategies aimed at delivering growth factors to promote skin wound healing. The approaches involving transient delivery of keratinocyte growth factor (KGF) to wounds performed in the engrafted human skin included (1) KGF gene transfer by intradermal adenoviral injection; (2) KGF gene transfer by adenoviral vector immobilized in a fibrin carrier; and (3) KGF-adenoviral gene-transferred human fibroblasts embedded in a fibrin matrix. All delivery systems achieved KGF protein overproduction at the wound site, with a concomitant re-epithelialization enhancement. However, although direct gene delivery strategies exhibited variability in terms of the number of successfully transduced humanized mice, the use of genetically modified fibroblast-containing matrix as an in situ protein bioreactor was highly reproducible, leading to a significant improvement of the overall healing process. This latter approach appeared to be the most reliable means to deliver growth factors to wounds and also avoided the potential danger of scoring cases of faulty administration as therapeutic failures and direct exposure to viral vectors. The combined use of cell and gene therapy appears a robust tool to aid healing in a clinical context.

Neutrophils (PMN), potent phagocytes, are the first line of the host immune defence against microorganisms, especially bacteria. Their half-life is very short and they are eliminated through apoptosis. Delayed neutrophil apoptosis is a characteristic feature of human osteomyelitis arising from Gram-negative or Gram-positive bacterial infection. The aim of this study was to investigate the modulation of apoptosis during infection of the human neutrophils by Staphylococcus aureus or Escherichia coli, the most common isolate in osteomyelitis. Analysis of host cells by flow cytometry using propidium iodide or annexin V labelling revealed an apoptosis inhibition after bacterial infection or treatment with LPS or LTA. We detected the secretion of cytokines such as IL-6, TNF-alpha and IL-1 beta by infected neutrophils. The addition of monoclonal antibodies to each cytokine abolished the protection against apoptosis. The anti-apoptotic Bcl-x(L) protein expression was increased and the pro-apoptotic Bax-alpha protein expression was decreased. These results identify a novel apoptotic effect in bacteria-infected cells that is mainly dependent on auto-production of cytokines and is correlated with Bax-alpha/Bcl-x(L) ratio. This may be a mechanism through which to resolve bacterial osteomyelitis infection.

Based on the well established involvement of IL-1beta in inflammatory hyperalgesia, we have assessed the possible role played by IL-1beta in a murine model of bone cancer-induced pain. With this aim, we measured IL-1beta levels at the region of the tibia and the spinal cord in mice bearing a tibial osteosarcoma induced by the inoculation of NCTC 2472 cells, and we tested whether the IL-1 receptor antagonist, anakinra, inhibits some hypernociceptive reactions evoked by the neoplastic injury. Parallel experiments were performed in mice with a chronic inflammatory process (intraplantar injection of complete Freund's adjuvant, CFA). IL-1beta levels were increased in the tibial region of osteosarcoma-bearing mice and in the paws of inflamed mice. To a lesser extent, the content of IL-1beta in the spinal cord was also augmented in both situations. Osteosarcoma-induced thermal hyperalgesia was inhibited by 30 and 100 mg/kg of systemic anakinra, but only 300 mg/kg prevented inflammatory thermal hyperalgesia. Mechanical hyperalgesia induced by the osteosarcoma was blocked by 100 and 300 mg/kg of anakinra, whereas a partial reversion of inflammatory mechanical hyperalgesia was induced by 300 mg/kg. Anakinra, intrathecally administered (1 and 10 microg) did not modify hyperalgesia of either origin. Besides, both tumoral and inflammatory mechanical allodynia remained unaltered after the administration of anakinra. In conclusion, some hyperalgesic symptoms observed in this model of bone cancer are mediated by the peripheral release of IL-1beta and may be inhibited by antagonists of type I IL-1 receptors with a similar or greater potency than symptoms produced by inflammation.

Cytokines generate nitric oxide (NO) in osteoblasts and neutrophils through the induction of NO synthase isoforms, endothelial (NOS3) and inducible (NOS2), thereby producing bone loss. In osteomyelitis (OM), a chronic infection of the bone, homozygosity for the NOS3 (27-bp repeat, intron 4 polymorphism) 4 allele was significantly more frequent among the 80 patients than in 300 healthy controls ( p = 0.044). No significant differences were found for other polymorphisms of the NOS genes such as NOS3 , the promoter (−786T/C), and the missense change (E298D) in exon 7, and for NOS2 , the G/A substitution at position 37498 in exon 22, the (CCTTT) n , and (TAAA) n micro-satellites and the −954G/C in the promoter. Serum NO levels were significantly higher only in the OM patients homozygous for the NOS3 (27-bp repeat, intron 4 polymorphism) 4 allele, compared to controls. In the presence of bacteria or bacterial products, the neutrophils of these patients produced more NO. However, immunolabelling of osteoblasts for NOS3 in biopsy tissues did not correlate with the carriage of a determined NOS polymorphism but with the presence of bone inflammation. This is the first report of an association between a NOS3 polymorphism and the risk of developing OM.

The aim of this study was to develop a new keratinocyte culture system on a dermal equivalent suitable for skin wound closure. Our dermal matrix is based on a fibrin gel from plasma cryoprecipitate containing live human fibroblast (from human foreskin). Keratinocytes obtained from primary culture according to the Rheinwald and Green method, were seeded on the gel at different seeding ratios. In all cases, the keratinocytes plated on the dermal equivalent grew to confluence and stratified epithelium was obtained within 10-15 days in culture. Early expression of basal membrane proteins was detected by immunostaining with laminin and type IV collagen antibodies. Cell proliferation was detected both in the epidermal layer and in the fibroblast embedded in the gel as assessed by BrdU incorporation. Detachment of composite cultures from dishes or flasks is a simple and quick procedure without the need for dispase treatment. Grafting of composite cultures to nude mice gave rise to an orderly stratified, orthokeratinized epithelium resembling human epidermis. A number of advantages including a large expansion factor without the need of 3T3 feeder layer, the availability of fibrin/plasma cryoprecipitate from blood banks and the versatile manipulation of composite cultures suggest that this system could be suitable for the definitive coverage of severely burned patients.

Aim: We analyzed retrospectively data about treatment outcome with existing treatments in DTF. Methods: Descriptive analysis of data related to diagnosis and treatment from all DTF was collected in patients (p) between Sept. 1999 and Nov. 2013 in 26 hospitals of GEIS . Ethics committee approval was obtained. Results: 185 patients. Age: median 37 years (6-85) .63.2% female .Median time lapse from first symptom to diagnosis: 4 months(m). Location : trunk wall 81p (43.8%), extremities 50p (27%), retroperitoneum 10p (5.4%), Gastro-intestinal 16p (8.6%), Head/neck 6p( 3.2%), others 6p(3.2%), 2p (1.1%) missing . 20p (11%) presented a second neoplasia (throughout the whole process). Median tumor size: 8 cm(range 1-96). 3 p (1.6%) presented distant peritoneal disease. First treatment : No treatment 6p (2.7%), Surgery (S) alone 144 p ( 77.8%, 3p >1), S+radiotherapy (RT) 7p (3.7%), S+ chemotherapy (CT) 2 p (1.08%), S+hormonetherapy (HT) 3p (1.62%), S +HT+ Non steroidal antiinflammatory drug (NSAID)+Tirosin-kinasa inhibitor(TKI) 1p (0.54%), CT 5p (2.7%), RT 3p ( 1.6%),NSAID 5p (2.6%), HT 2p (1.08%), TKI 1p(0.54%), HT+NSAID 6p (2.7%). 128p (69.2%) became free of disease, in 49p (26.5%) residual disease remained . 61p (33%) progressed. Median Progression free survival (PFS) :109 m (95%CI 44.6-175.1).Multivariate analysis for PFS: extended/radical surgery(p=0.000) and tumor size Response to CT /other treatments CT treatment Other treatments (TKI, HT, NSAID) p % p % PR 12 30.7 4 7.4 SD 15 38.4 31 55.3 PD 3 7.6 10 17.8 NE 4 10.2 6 10.7 NA 5 12.8 5 8.9 Total 39 100 56 100 PR: partial response; SD: stable disease; PD: progressive disease; NE: non evaluable; NA: non available data. Conclusions: Surgical quality and tumor size seems to play a relevant role in predicting PFS in DFT. Other sistemic treatments showed meaningful activity in progressive disease. Disclosure: All authors have declared no conflicts of interest.

Aim: SFT are soft tissue sarcomas with low incidence and intermediate biological potential behavior. The main aim of this observational study is to get new insights in clinical presentation, biological behavior and therapeutic approaches of SFT in our country. Methods: A web-based registry was built to collect diagnostic and therapeutic processes in patients (p) with SFT diagnosed in 22 centers (centers of reference and secondary centers) involved in this program. Median progression-free survival (PFS) and overall survival (OS), were estimated by Kaplan-Meier method. Ethics committee approval was obtained. Results: Between September 1999 and November 2013, 163 p (median age 52 years, 52.8% females, 88% had Karnofsky≥80%) were diagnosed. 12 p lacked some relevant data after diagnosis. 7 hospitals included 70% of cases. Primary tumor location was: 24% extremities and trunk-wall, 15% lungs, 13% pleural, 13% head and neck, 14% retroperitoneum and 2.5% meningeal sites. Excisional biopsy and core-biopsy were the method of diagnosis in 65% and 22% of p respectively. Median size was 8.6 cm (range 1-29). 11 p were metastatic at diagnosis. Initial treatment was surgery in 89% of 151 p, followed by adjuvant treatment in 23: 2 chemotherapy (CT), 15 radiotherapy (RT), 5 CT and RT, and 1 radiosurgery. The remaining p received: 1 CT, 2 RT, 1 sunitinib and 2 palliative care. 36 p progressed: 19 p could be resected. Antiangiogenic therapy was used in advanced disease:13 p received sunitinib (5 first line, 3 second line and 5 further lines), 5 p temozolamide-bevacizumab (3 first line and 2 further lines) and 2 p pazopanib (1 first line) . Sunitinib showed clinical benefit (CB) in 8 cases (35, 36, 30, 22, 8, 5, 4 and 2 months of duration), 4 progressions (PR) and 1 unknown. 2 p stopped for toxicity grade III-IV. Temozolamide-bevacizumab experienced CB in 3 cases (30,12, and 6 months of duration), 1 PR and 1 p stopped for toxicity grade III. 1p progressed with pazopanib and 1 was not evaluated for response. Median PFS was 5 years and median OS was not reached. OS and PFS at 10 years were 70% and 50% respectively. Conclusions: SFT are tumors that appear in different localizations, where radical surgery is the treatment of choice. In advanced disease, antiangiogenic therapy could provide longer CB in 60% of progressing cases. Studies with these drugs in advanced SFT are guaranteed. Disclosure: All authors have declared no conflicts of interest.