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Primary responses in sepsis-mediated inflammation are regulated by pro-inflammatory cytokines. Variations in the cytokine genes might modify their transcription or expression, plasma cytokines levels and response to sepsis. Activation protein-1 (AP-1) and NF-κB regulate cytokines gene expression in sepsis. A total of 90 severely septic and 91 non-infected patients were prospectively studied. IL-1α ( –889 C/T), IL-1β ( +3954 C/T), IL-6 ( –174 G/C), TNF-α ( –238 G/A), TNF-α ( –308G/A), IL-8 ( –251A/T) and IL-10 ( –1082 G/A) SNPs, plasma IL-1β, IL-4, IL-6, IL-8, IL-10, IL-13, IFN-γ, TNF-α and monocyte chemoattractant protein 1 (MCP-1) levels, and AP-1 and NF-κB gene expression by neutrophils were assessed. A allele carriers of TNF-α ( –238 G/A) SNP were less frequent among septic patients. IL-6, IL-8, IL-10, TNF-α and MCP-1 levels were higher, and AP-1 and NF-κB gene expressions lower in septic patients. Sepsis was independently associated with higher fibrinogen, neutrophils counts and IL-8 levels, lower prothrombin, absence of the variant A allele of the TNF-α (–238 G/A) SNP, and haemodynamic failure. Death was independently associated with a higher APACHE II score, higher IL-8 levels, and the diagnosis of sepsis. TNF-a ( –238 G/A) SNP could protect against sepsis development. Higher IL-8 levels are predictive of sepsis and mortality.

Osteoradionecrosis is one of the most severe complications of radiotherapy administered for head and neck tumors. We present the first two cases of advanced and refractory mandibular osteoradionecrosis treated by application of a novel autologous cross-linked 3D serum matrix. Patients were followed clinically and radiographically up to 24 months. Complete wound healing and intact mucosal cover were achieved in both cases. At 12 months, the radiographic values showed an almost complete regeneration of the bone defect, which continued a favourable progression increased to the maximum by 24 months after surgery. The use of an autologous serum-derived scaffold proved to be a quick, predictable, cost-effective and safe adjunct to the conservative surgical treatment of this pathology.

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.

To restore corneal transparency and vision loss after an injury on the ocular surface, the use of human stem cells from different origins has been recently proposed. Mesenchymal stem cells (MSCs) seem to be an appropriate adult source of autologous stem cells due to their accessibility, high proliferation rate, and multipotent capacity. In this work, we developed a simple culture system to prepare a graft based on a fibrin membrane seeded with human MSCs. A commercial kit, PRGF Endoret®, was used to prepare both, the growth factors used as culture media supplement and the fibrin membrane grafts. Adipose-derived MSCs (Ad-MSCs) were expanded, characterised by flow cytometry and their multilineage differentiation potential confirmed by inducing adipogenesis, osteogenesis and chondrogenesis. Ad-MSCs seeded on the fibrin membranes were grafted onto athymic mice showing good biocompatibility with no adverse reactions observed during the follow up period. These findings support the assumption that a system in which all the biological components (cells, grow factors and carrier) are autologous, could potentially be used for future ex vivo expansion of Ad-MSCs to treat ocular conditions such as an inflammatory milieu, traumatic scars and loss of the regenerative capacity of the corneal epithelium that compromise the quality of vision.

Background and importance Sutures to replace tissue adhesives have enhanced importance. However, commercialised drugs are allogenic, synthetic and expensive, increasing surgery costs. Aim and objectives To produce an autologous tissue adhesive (ATA) easily compounded in ophthalmological surgery. To show evidences of the safe and effectiveness of the ATA in preclinical studies. Material and methods To produce 4 mL of ATA based on a fibrinogen (FC) and thrombin concentrate (TC) (proportion1:1), 20 mL of donor blood plasma were precipitated with protamine to prepare FC, and then 20 mL of plasma were precipitated with acetic acid to obtain a TC in a buffer (CaCl2, NaHCO3, NaCl). Drug was conditioned in two 2 mL syringes for topical ophthalmic administration by mixing with a needle. The in vitro toxicity of the drug was studied in a human corneal epithelial model (described as QobuR), to evaluate the grade of irritation after 30 min of exposition time.1 Pterygium surgery was performed in four eyes of white New Zealand rabbits, using ATA to fix a frontal conjunctival autograft (4×5 mm) into the temporal bulbar conjunctive. The grafted eyes were evaluated in vivo by clinical evaluation for 14–28 days and ex vivo by histology. Results ATA produced from each donor showed a mean of 18.0 g/L of fibrinogen and 1500 UI/mL of thrombin. ATA instantly produced homogeneous clots when it was mixed with a needle. Three in vitro studies of four ATA showed non-irritation due to high survival cell viabilities (>80%). Good preclinical results were found: 20 mm2 autograft could be fixed successfully. Time for complete tissue adhesion was minimal (3–5 min). Inflammation and adverse events were absent in all cases. The prospective clinical evaluation was positive for follow-up in all cases and included integration and vascularisation of the grafts. Histology supported the in vivo evidence. Staining of the autograft section showed inner vessels and the regeneration of the surrounding conjunctive tissue. Conclusion and relevance It is possible to compound an ATA easily from whole blood, in a hospital pharmacy, for ophthalmological surgery, where the necessary volume is very low. This ATA was safe and effective, supported by our preclinical studies. This ATA could allow the possibility of replacing the suture in surgery with a low cost drug. References and/or acknowledgements 1. https://www.ncbi.nlm.nih.gov/pubmed/30690064 No conflict of interest.

AimTo investigate the difference in adhesion and rebubbling rate between eye bank and surgeon prepared Descemet membrane endothelial keratoplasty (DMEK) tissues.MethodsLaboratory and clinical retrospective comparative interventional case series. Research corneal tissues were obtained for laboratory investigation. The clinical study involved patients with endothelial dysfunction who underwent DMEK surgery and tamponade with air. Tissues were stripped using a standard DMEK stripping technique (SCUBA) and shipped as prestripped or loaded in a 2.2 intra-ocular lens cartridge with endothelium facing inwards (preloaded) before transporting from the eye bank to the surgeon. For surgeon prepared tissues, all the grafts were stripped in the theatre and transplanted or stripped in the laboratory and tested immediately. Adhesion force and elastic modulus were measured in the centre and mid-periphery in a laboratory ex vivo investigation using atomic force microscopy, while rebubbling rates were recorded in the clinical study.ResultsThere was no difference in endothelial cell viability between surgeon or eye bank prepared tissue. Surgeon-stripped DMEK grafts in the laboratory investigation showed significantly higher elastic modulus and adhesion force compared to prestripped and preloaded tissues (pConclusionsDecreased adhesion forces and elastic modulus in eye bank prepared tissues may contribute to increased rebubbling rates.

Author(s): Perez-Is, Laura; Ocana, Marcos G; Montes, A Hugo; Carton, Jose A; Alvarez, Victoria; Meana, Alvaro; Fierer, Joshua; Valle-Garay, Eulalia; Asensi, Victor | Abstract: BACKGROUND:Osteomyelitis is a bone infection, most often caused by Staphylococcus aureus, in which neutrophils play a key role. Cathepsin G (CTSG) is a bactericidal serine protease stored in the neutrophil azurophilic granules. CTSG regulates inflammation, activating matrix metalloproteinases (MMPs), and coagulation. Lactoferrin (LF), a neutrophil glycoprotein, increases CTSG catalytic activity and induces inflammation. The aim of this study was to analyze a potential association between a CTSG gene polymorphism (Asn125Ser or N125S, rs45567233), that modifies CTSG activity, and could affect susceptibility to, or outcome of, bacterial osteomyelitis. METHODS:CTSG N125S polymorphism was genotyped in 329 osteomyelitis patients and 415 controls), Blood coagulation parameters, serum CTSG activity, LF, MMP-1, MMP-13, and soluble receptor activator for nuclear factor κ B ligand (sRANKL) levels were assessed in carriers of the different CTSG genotypes. RESULTS:CTSG N125S (AG) genotype was significantly more frequent among osteomyelitis patients than controls (15.5% vs. 9.4%, p = 0.014). CTSG N125S variant G allele (AG +GG) was also more frequent among osteomyelitis patients (8.1% vs. 4.7%, p = 0.01). Serum CTSG activity and LF levels were significantly higher in osteomyelitis patients carrying the G allele compared to those with the AA genotype, (pl0.04). Serum MMP-1 was lower in the G allele carriers (p = 0.01). There was no association between these genotypes and clinical characteristics of osteomyelitis, or coagulation parameters, MMP-13, and sRANKL serum levels. CONCLUSIONS:Differences in the CTSG gene might enhance osteomyelitis susceptibility by increasing CTSG activity and LF levels.

Non-healing chronic skin ulcers are considered a major biological, psychological, and financial burden for both patients and health systems. Multidisciplinary endeavors are required to address this refractory disease, in order to find definitive solutions that lead to improved living conditions. Diabetes, venous stasis, arterial insufficiency, pressure and radiation are common risk factors associated with chronic wounds. Unfortunately, the cured state for these wounds has a high relapse rate, which adversely affects the patient’s quality of life. Nevertheless, advances on regenerative medicine have allowed the development of cell-based therapies that promote wound healing by increasing cell migration and differentiation. Particularly, mesenchymal stem cells (MSCs) and their acellular derivatives have emerged as an attractive therapeutic agent in various diseases, including chronic skin ulcers, due to their role in immunomodulation and tissue regeneration. In this review discusses the characteristics of MSCs as well as their regenerative properties and their action mechanisms on wound healing. Finally, the perspectives of MSCs and their acellular derivatives in clinical chronic skin ulcer therapy are also explored.

Many types of soft tissue grafts have been used for the reconstruction of oral mucosal defects. The best results are achieved with mucosal grafts; however, when large areas must be grafted, sufficient donor tissue is not available. Tissue engineering represents an alternative method to obtain sufficient autologous tissue for reconstructing oral wounds. Herein we present a pediatric patient with hemifacial microsomia and congenital ankyloglossia requiring multiple surgical interventions, and in which an autologous full-thickness tissue-engineered oral mucosa was used for successful oral reconstruction. Our study demonstrates that even under challenging conditions, robust tissue-engineered products, such as the fibrin-based oral mucosa described here, can achieve successful tissue regeneration.

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.

Summary Background Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in COL7A1. The clinical manifestations are highly variable from nail dystrophy to life-threatening blistering, making early molecular diagnosis and prognosis of utmost importance for the affected families. Mutation identification is mandatory for prenatal testing. Objectives To conduct the first mutational analysis of COL7A1 in a Spanish cohort, to assess mutation consequences at protein/mRNA level and to establish genotype–phenotype correlations. Methods Forty-nine Spanish patients with DEB were studied. Antigen mapping was performed on patient skin biopsies. COL7A1 mutation screening in genomic DNA was performed by polymerase chain reaction (PCR) and direct sequencing. Mutation consequences were determined by reverse transcriptase-PCR. Results Eight patients belonged to three unrelated families with dominant DEB. Forty-one were affected with recessive DEB (RDEB). Specifically, 27 displayed the severe generalized subtype, eight the other generalized subtype and six a localized phenotype (two pretibial, three acral and one inversa). Thirty-five mutations were identified, 20 of which are novel. The pathogenic mutation c.6527insC accounted for 46·3% of Spanish RDEB alleles. A consistent genotype–phenotype correlation was established. Conclusions Although the COL7A1 database indicates that most DEB mutations are family specific, the pathogenic mutation c.6527insC was highly recurrent in our cohort. This level of recurrence for a single genetic defect has never previously been reported for COL7A1. Our findings are essential to the clinicians caring for patients with DEB in Spain and in the large population of Spanish descendants in Latin America. They also provide geneticists a molecular clue for a priority mutation screening strategy.

The transplant of pancreatic islets into the liver can restore normal blood glucose levels in patients with type I diabetes. However, long-term results have indicated that the site and method of transplantation still need to be optimized to improve islet engraftment. This study was designed to assess the efficiency of the use of clotted blood plasma containing fibroblasts ("plasma-fibroblast gel") as a scaffold for subcutaneous islet transplantation in diabetic athymic mice. Islets embedded in the plasma-fibroblast gel were able to resolve hyperglycemia in transplanted mice, restoring normoglycemia over a 60-day period and allowing gradual body weight recovery. Glucose clearances were significantly improved when compared to those recorded in diabetic animals and similar to those observed in the control group (free islets transplanted beneath the kidney capsule). Histological evaluation revealed functional islets within a subcutaneous tissue rich in collagen fibers that was well vascularized, with blood vessels observed around and inside the islets. These findings suggest that this approach could be used as an alternative option for the treatment of type I diabetes in human clinical practice.

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.

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.

AIM The aim of this study was to evaluate effective bone regeneration using an autologous serum scaffold (alone or seeded with autologous bone marrow-mesenchymal stem cells, BM-MSCs), when implanted in a 30 mm length segmental mandibular defect in sheep. MATERIALS AND METHODS The bone defect was filled either with serum scaffold alone (control group; n = 5) or combined with BM-MSCs (experimental group; n = 10). Bone regeneration was determined at 12 (T12; 2 control sheep and 4 experimental sheep) and 32 weeks (T32; 3 control and 6 experimental sheep), as measured by computed and microcomputed tomography and histological examination. RESULTS Two sheep of the Experimental group died after surgery. While complete bone union in the control group was only observed at T32, it was observed both at T12 (1/4 sheep) and T32 (3/4 sheep) in the experimental group. When properties/characteristics of new bone where compared, a better bone quality, similar to native bone, was observed in the scaffold combined with BM-MSCs. CONCLUSIONS Based on these results, we conclude that the serum scaffold can promote efficient repair of large bone defects, but the combination with BM-MSCs accelerates this process, increasing significantly the amount and quality of bone formed.

Introduction: An artificial complete skin (dermis and epidermis) model has been developed in the Tissue engineering unit of the Centro Comunitario de Sangre y Tejidos del Principado de Asturias (CCST) and CIEMAT. This engineered skin has been employed for the treatment of severe epithelial injuries. In this paper, the clinical results obtained with this engineered skin during the last 18 months were evaluated. Patients, material and methods: (a) Culture: Cells (fibroblasts and keratinocytes) were obtained from biopsies by a double enzymatic digestion. After an expansion period, fibroblasts were seeded in an artificial dermis based on human plasma. Keratinocytes were seeded over this dermal surface. (b) Patients: 20 skin biopsies were processed (13 burned patients, 5 giant nevus, 1 GVHD, 1 neurofibromatosis), which came from different hospitals across the country. About 97,525 cm2 of engineered skin were cultured. Results: The engineered skin took in all patients. The take percentage depended on previous pathology (burned patients 10–90%; non-critical patients 70–90%). The epithelization obtained was permanent in all cases. During the follow-up period, epithelial loss, blistering injuries or skin retractions were not observed. The aesthetic and functional results were acceptable. Conclusions: This artificial skin has demonstrated to be useful for the definitive treatment of patients with severe skin injuries. This work shows that it is possible to produce this prototype in an hospitalarian laboratory and distribute it to different hospitals across the country.

We report a rare case of a male patient without known immunodeficiency consecutively diagnosed with visceral leishmaniasis, brain abscess and cavitating pneumonia in the 3rd decade of life. Chronic granulomatous disease (CGD) was diagnosed by a nitroblue tetrazolium test. A p47-phox mutation of the NADPH oxidase of the leukocytes was suspected by immunoblotting and confirmed by DNA analysis. The patient was homozygous for this mutation while his mother and sister were heterozygous asymptomatic carriers. After the CGD diagnosis the patient started a chronic prophylactic regimen with subcutaneous interferon-gamma (0.05 mg/m2 of body surface/three times a week), and oral trimethoprim-sulfamethoxazole and itraconazole (both at 5 mg/kg/day) with no subsequent infections after 12 months of follow-up.

The radial forearm free flap has been popular in many areas of reconstructive surgery. Despite the many attributes of this flap in maxillofacial reconstruction, one of the disadvantages has been the morbidity of the donor site. Allogeneic cultured epidermis has been successfully applied on large second degree burns and on chronic leg ulcers. Autologous human keratinocytes and fibroblast equivalents can be cultured in-vitro from a small skin sample in order to produce a sufficient amount of epithelial autografts to cover the large defects of third-degree burn wounds. Interestingly, transplanted cultured epidermis retains characteristics of the original donor site. We report a case of a patient who underwent skin replacement by cultured epithelial autograft after wound breakdown occurred in the forearm donor site during the early postoperative period. This method could represent an auspicious alternative to conventional grafting methods for forearm free flap reconstruction. To the best of our knowledge, skin replacement by cultured epithelial autografts in this region has not been extensively described in the literature.

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.

Background. Coagulation and fibrinolysis are important in infections and systemic inflammatory response syndrome. Polymorphisms in plasminogen activator inhibitor-1 (PAI-1, SERPINE1) and tissue plasminogen activator (tPA, PLAT), such as PAI-1 (-675 4G/5G deletion/insertion) and tPA (Alu insertion/deletion [I/D]), are associated with strokes, myocardial infarctions, bacterial infections and septic shock severity, and trauma. Osteomyelitis is a mostly posttraumatic, Staphylococcal bone infection. Patients and methods. tPA Alu (I/D) (rs4646972) and PAI-1 (4G/5G) (rs1799889) polymorphisms were studied by DNA amplification with polymerase chain reaction in 261 patients with osteomyelitis and in 299 matched blood donors. Plasma PAI-1/tPA complex was assessed by enzyme-linked immuosorbent assay. Results. II homozygotes (37.9% vs 19.1%) and I allele carriers (56.3% vs 46.3%) for the tPA Alu (I/D) polymorphism were significantly more frequent in osteomyelitis patients compared to controls (P< .001). II genotype carrier osteomyelitis patients had lower PAI-1/tPA complex levels compared to those with theD allele (P ≤.04). There was no association between these genotypes and chronicity of osteomyelitis, post-traumatic etiology, or with a specific bacterial etiology. PAI-1 (4G/4G) homozygotes were not significantly different between osteomyelitis patients and controls (P= .1). Conclusions. We report for the first time to our knowledge an association between the tPA Alu (I/D) polymorphism and susceptibility to bacterial osteomyelitis, perhaps by fibrinolysis dysfunction.

Cutaneous diabetic wounds greatly affect the quality of life of patients, causing a substantial economic impact on the healthcare system. The limited clinical success of conventional treatments is mainly attributed to the lack of knowledge of the pathogenic mechanisms related to chronic ulceration. Therefore, management of diabetic ulcers remains a challenging clinical issue. Within this context, reliable animal models that recapitulate situations of impaired wound healing have become essential. In this study, we established a new in vivo humanised model of delayed wound healing in a diabetic context that reproduces the main features of the human disease. Diabetes was induced by multiple low doses of streptozotocin in bioengineered human-skin-engrafted immunodeficient mice. The significant delay in wound closure exhibited in diabetic wounds was mainly attributed to alterations in the granulation tissue formation and resolution, involving defects in wound bed maturation, vascularisation, inflammatory response and collagen deposition. In the new model, a cell-based wound therapy consisting of the application of plasma-derived fibrin dermal scaffolds containing fibroblasts consistently improved the healing response by triggering granulation tissue maturation and further providing a suitable matrix for migrating keratinocytes during wound re-epithelialisation. The present preclinical wound healing model was able to shed light on the biological processes responsible for the improvement achieved, and these findings can be extended for designing new therapeutic approaches with clinical relevance. This work was supported by grants from the Science and Innovation Ministry of Spain (SAF2010-16976), from the European VI Framework Programme (LSHB-CT-512102), from Comunidad de Madrid (S2010/BMD-2420; CELLCAM) and from Fundacion Ramon Areces (CIVP16A1864).

Corneal keratoplasty (penetrating or lamellar) using cadaveric human tissue, is nowadays the main treatment for corneal endotelial dysfunctions. However, there is a worldwide shortage of donor corneas available for transplantation and about 53% of the world's population have no access to corneal transplantation. Generating a complete cornea by tissue engineering is still a tough goal, but an endothelial lamellar graft might be an easier task. In this study, we developed a tissue engineered corneal endothelium by culturing human corneal endothelial cells on a human purified type I collagen membrane. Human corneal endothelial cells were cultured from corneal rims after corneal penetrating keratoplasty and type I collagen was isolated from remnant cancellous bone chips. Isolated type I collagen was analyzed by western blot, liquid chromatography -mass spectrometry and quantified using the exponentially modified protein abundance index. Later on, collagen solution was casted at room temperature obtaining an optically transparent and mechanically manageable membrane that supports the growth of human and rabbit corneal endothelial cells which expressed characteristic markers of corneal endothelium: zonula ocluddens-1 and Na+/K+ ATPase. To evaluate the therapeutic efficiency of our artificial endothelial grafts, human purified type I collagen membranes cultured with rabbit corneal endothelial cells were transplanted in New Zealand white rabbits that were kept under a minimal immunosuppression regimen. Transplanted corneas maintained transparency for as long as 6 weeks without obvious edema or immune rejection and maintaining the same endothelial markers that in a healthy cornea. In conclusion, it is possible to develop an artificial human corneal endothelial graft using remnant tissues that are not employed in transplant procedures. This artificial endothelial graft can restore the integrality of corneal endothelium in an experimental model of endothelial dysfunction. This strategy could supply extra endothelial tissue and compensate the deficit of cadaveric grafts for corneal endothelial transplantation.

We have previously shown that stimulation of peripheral opioid receptors by exogenous opiates counteracts the thermal hyperalgesia elicited by a tibial osteosarcoma due to intraosteal inoculation of NCTC 2472 cells to mice. Aiming to study whether pheripheral endogenous enkephalins could also counteract this painful symptom, we assayed in this model the effects of PL37, an orally active dual inhibitor of enkephalin inactivating enzymes. Oral administration of PL37 (25 mg/kg) completely supressed osteosarcoma-induced thermal hyperalgesia through the activation of micro-opioid receptors, since the administration of cyprodime (1 mg/kg) inhibited its antihyperalgesic effect. Neither naltrindole (0.1 mg/kg) nor nor-binaltorphimine (10 mg/kg) modified this PL37-induced antihyperalgesic effect. Moreover, the inhibition of the antihyperalgesic effect induced by PL37 after the administration of naloxone-methiodide (2 mg/kg), a non selective opioid antagonist that does not cross the blood-brain barrier, demonstrates the involvement of peripheral opioid receptors. In contrast, centrally mediated effects may be detected when assaying a higher dose of PL37 (50 mg/kg). Besides, the administration of gabapentin (6.25-25 mg/kg, i.p.) dose-dependently inhibited osteosarcoma-induced thermal hyperalgesia. Interestingly, the combined administration of subeffective doses of PL37 and gabapentin completely prevented this type of thermal hyperalgesia. An isobolographic analysis of this interaction demonstrated a synergistic interaction between both drugs.

Transient thermal, but not mechanical, hypoalgesia appears at the early stages of the development of an hyperalgesic murine osteosarcoma. This hypoalgesia is suppressed by the administration of naloxone, its peripherally acting analog naloxone methiodide, the mu- and delta-opioid receptor antagonists cyprodime and naltrindole, or the CRF receptor antagonist, alpha-helical CRF (9-41). When immunohistochemical assays were performed with an anti-beta-endorphin antibody, whose in vivo administration suppressed the analgesia, labeled mononuclear immune cells appeared both inside and surrounding the tumoral tissue. In conclusion, the peripheral action of beta-endorphin, released in response to the osteosarcoma seems responsible for the observed thermal analgesia.

The intratibial inoculation of NCTC 2472 cells induces an osteosarcoma in C3H/HeJ mice. These mice show thermal hyperalgesic responses which may be blocked by the local administration of opiates over the tibial tumoral mass ( Menendez L, Lastra A, Hidalgo A, Meana A, Garcia E, Baamonde A. Peripheral opioids act as analgesics in bone cancer pain in mice. NeuroReport 2003b;14:867–9 ). The aim of this report was to characterize the analgesic responses obtained by activating peripheral opioid receptors in bone cancer pain. Here, we initially describe that this osteosarcoma induces mechanical as well as thermal hyperalgesia. Loperamide, an opioid agonist unable to cross the blood–brain barrier, inhibits both thermal and mechanical hyperalgesia when s.c. injected, locally over the tibial tumoral mass (7.5–75 μg) or distantly, under the fur of the neck (4 mg/kg). These analgesic effects seem peripherally mediated since they are reverted by the administration of naloxone methiodide (10 mg/kg) and because the withdrawal latencies of the contralateral, non-affected, paws remain unaltered. Furthermore, only cyprodime (1 mg/kg) but not naltrindole (0.1 mg/kg) or nor-binaltorphimine (10 mg/kg) blocked these effects, showing the involvement of μ-opioid receptors in the peripheral analgesia induced by loperamide on thermal and mechanical hyperalgesia. The advantages of using peripheral acting opiates – devoid of central colateral effects – for the treatment of cancer related pain are suggested.

Previous reports have shown that systemic administration of morphine can prevent the thermal hyperalgesia induced by the development of an osteosarcoma in C3H/HeJ mice after the implantation of NCTC 2472 cells. We show here that this type of hyperalgesia is also abolished by the local administration of morphine given at low doses (10 nmol), or the peripheral acting opioid receptor agonist loperamide (146 nmol). The analgesic effect of loperamide is prevented by the administration of the opioid receptor antagonist naloxone methiodide (10 mg/kg, i.p.), which is unable to cross the blood-brain barrier. These results provide evidence which supports the fact that peripheral opioids could be useful tools in the management of some types of cancer pain.

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.

Cutaneous wound-healing disorders are a major health problem that requires the development of innovative treatments. Whithin this context, the search for reliable human wound-healing models that allow us to address both mechanistic and therapeutic matters is warranted. In this study, we have developed a novel invivo wound-healing model in a genetically modified human context. Our model is based on the regeneration of human skin on the back of nude mice by transplantation of a cultured bioengineered skin equivalent previously designed in our laboratory. In this setting, human keratinocytes in the epidermal compartment were genetically modified with a retroviral vector encoding the enhanced green fluorescent protein (EGFP). After stable engraftment of the EGFP skin was achieved (9-12 wk after grafting), a small circular full thickness wound was performed on this mature human skin. A wide variety of parameters involved in wound healing were monitored, including tissue architecture, cell proliferation, epidermal differentiation, dermal remodelling, and basement membrane regeneration. Wounded gene-targeted skin-humanized mice re-capitulated native skin wound-healing features. In addition, when keratinocyte growth factor (KGF), a growth factor that has been shown to improve wound healing, was added to wounds during 3 d, the re-epithelialization was significantly accelerated. The present wound-healing model system provides a suitable in vivo tool to test gene transfer strategies for human skin repair. It also serves as a complementary platform for studies in genetically modified mice and as a model to evaluate pharmaceutical therapeutic approaches for impaired wound healing.