Experimental study of the application of a new bone cement loaded with broad spectrum antibiotics for the treatment of bone infection. / Estudio experimental de la aplicación de un nuevo cemento óseo cargado con antibióticos de amplio espectro para el tratamiento de la infección ósea.

OBJECTIVES: To evaluate the in vivo behaviour of a new bone cement loaded with antibiotics, in a rabbit bone infection model. MATERIAL AND METHODS: Sixteen New Zealand rabbits divided into 4 groups were used, depending on the cement (commercial or experimental) and the antibiotic (vancomycin or linezolid) used to control a bone infection caused by Staphylococcus aureus. The commercial cement is Palacos® R and the experimental cement has been achieved by adding PLGA to the solid phase of Palacos® R cement. A novel histological staging method based on bone histoarchitecture has been used. This staging allows us a global vision of bone repair capacity, in the presence of modified cement, and also allows us to correlate the damage generated with the functionality of the tissue. RESULTS: The degree of bone destructuration found depended on the type of cement and antibiotic, and was higher in the groups with commercial cement than in the experimental group (P<.01) and in the groups with linezolid with respect to vancomycin (P=.04) The percentage of macrophages varied exclusively depending on the antibiotic used, and was higher in the vancomycin groups (P=.04). DISCUSSION: The development of new formulations of bone cement that release more, and more prolonged, new generation antibiotics such as linezolid, present an in vivo behaviour superior to commercial cement, respecting the bone structure. This behaviour would have a clinical implication in fighting infections by increasingly resistant germs in the treatment of prosthetic infection.

Skeletal muscle IL-15/IL-15Rα and myofibrillar protein synthesis after resistance exercise.

In vitro and in vivo studies described the myokine IL-15 and its receptor IL-15Rα as anabolic/anti-atrophy agents, however, the protein expression of IL-15Rα has not been measured in human skeletal muscle and data regarding IL-15 expression remain inconclusive. The purpose of the study was to determine serum and skeletal muscle IL-15 and IL-15Rα responses to resistance exercise session and to analyze their association with myofibrillar protein synthesis (MPS). Fourteen participants performed a bilateral leg resistance exercise composed of four sets of leg press and four sets of knee extension at 75% 1RM to task failure. Muscle biopsies were obtained at rest, 0, 4 and 24 hours post-exercise and blood samples at rest, mid-exercise, 0, 0.3, 1, 2, 4 and 24 hours post-exercise. Serum IL-15 was increased by ~5.3-fold immediately post-exercise, while serum IL-15Rα decreased ~75% over 1 hour post-exercise (P<.001). Skeletal muscle IL-15Rα mRNA and protein expression were increased at 4 hours post-exercise by ~2-fold (P<.001) and ~1.3-fold above rest (P=.020), respectively. At 24 hours post-exercise, IL-15 (P=.003) and IL-15Rα mRNAs increased by ~2-fold (P=.002). Myofibrillar fractional synthetic rate between 0-4 hours was associated with IL-15Rα mRNA at rest (r=.662, P=.019), 4 hours (r=.612, P=.029), and 24 hours post-exercise (r=.627, P=.029). Finally, the muscle IL-15Rα protein up-regulation was related to Leg press 1RM (r=.688, P=.003) and total weight lifted (r=.628, P=.009). In conclusion, IL-15/IL-15Rα signaling pathway is activated in skeletal muscle in response to a session of resistance exercise.

Serum IL-15 and IL-15Rα levels are decreased in lean and obese physically active humans.

Circulating IL-15 presence is required to stimulate anti-adipogenic effects of the IL-15/IL-15Rα axis in adipose tissue. Although exercise increases blood IL-15 expression post-exercise, it remains inconclusive whether physical activity can alter the baseline concentrations of this cytokine. The aim of this study was to determine whether physical activity regulates circulating IL-15 and IL-15Rα in lean and obese individuals. Two hundred and seventy-six participants were divided into five groups according to physical activity (PA), body mass and type 2 diabetes mellitus (T2DM) diagnosis: (a) lean PA (N = 25); (b) lean non-PA (N = 28); (c) obese PA (N = 64); (d) obese non-PA (N = 79); and (e) obese non-PA with T2DM (N = 80). Serum IL-15 and IL-15Rα, blood glucose/lipid profile and body composition were measured. Serum IL-15 and IL-15Rα decreased in PA participants compared to non-PA (P < .05), while IL-15 and IL-15Rα increased in obese with T2DM compared to obese without T2DM (P < .05). No differences were observed between lean non-PA and obese PA. Serum IL-15Rα was associated with fasting glucose (R2 = .063), insulin (R2 = .082), HbA1c (R2 = .108), and HOMA (R2 = .057) in obese participants. Circulating IL-15 and IL-15Rα are reduced in lean and obese participants who perform physical activity regularly (≥180 min/week), suggesting a regulative role of physical activity on the circulating concentrations of IL-15 and IL-15Rα at baseline. Moreover, the relationship observed between IL-15Rα and glucose profile may indicate a role of the alpha receptor in glucose metabolism.

Development of advanced biantibiotic loaded bone cement spacers for arthroplasty associated infections.

The incidence increase of infections in patients with hip or knee implants with resistant pathogens (mainly some S. coagulase-negative and gram positive bacteria) demands advanced antibiotic loaded formulations. In this paper, we report the design of new biantibiotic acrylic bone cements for in situ delivery. They include a last generation antibiotic (daptomycin or linezolid) in combination with vancomycin and are performed based on a novel modification of the Palacos R® acrylic bone cement, which is based on two components, a liquid (methyl methacrylate) and a solid (polymeric phase). Hence, the solid component of the experimental formulations include 45wt% of microparticles of poly(D,L-lactic-co-glycolic) acid, 55wt% of poly(methyl methacrylate) beads and supplements (10wt-% each) of antibiotics. These formulations provide a selective and excellent control of the local release of antibiotics during a long time period (up to 2 months), avoiding systemic dissemination. The antimicrobial activity of the advanced spacers tested against S. aureus shows that single doses would be enough for the control of the infection. In vitro biocompatibility of cements on human osteoblasts is ensured. This paper is mainly focused on the preparation and characterization of cements and the studies of elution kinetics and bactericidal effects. Developed formulations are proposed as spacers for the treatment of infected arthroplasties, but also, they could be applied in other antibiotic devices to treat relevant bone-related infection diseases.

Long term behavior of biological prostheses used as abdominal wall substitutes.

BACKGROUND: Despite their degradation in the host organism, the benefits of collagen bioprostheses remain unclear. This study addresses the absorption and long-term host tissue incorporation of several collagen biomeshes. MATERIAL AND METHODS: Partial ventral hernial defects created in the abdominal wall of rabbits were repaired using the crosslinked meshes Permacol® or CollaMend®, or the non-crosslinked Surgisis®, Tutomesh® or Strattice®. After 90 and 180 days of implant, morphological studies and morphometric analysis of the thickness of the meshes were performed. Immunofluorescence confocal microscopy combined with differential interference contrast (DIC) imaging was used to distinguish newly formed collagen from that comprising the mesh. The macrophage response was examined by immunohistochemistry. RESULTS: At 90 days, the thinner non-crosslinked biomeshes Tutomesh and Surgisis were more fully degraded with much of their collagen replaced with loose connective tissue. By 180 days, both implants had been practically fully absorbed. In contrast, in Strattice only the outermost third was infiltrated by neoformed tissue. On both surfaces of the crosslinked meshes, a fibrous capsule with host cells lining its perimeter was observed at both time points, though at 180 days these cells had penetrated the mesh interior. At both implant times, Strattice showed the higher expression of collagen type I while collagen III expression was similar for all the meshes. The non-crosslinked materials elicited lower macrophage counts at both time points, significantly so for Strattice. The macrophage response decreased over time for all the meshes but Surgisis. CONCLUSIONS: Strattice, the thicker, more compacted non-crosslinked mesh showed the best balance between tissue incorporation and absorption while eliciting a minimal foreign-body reaction in the long-term.

Short-term behavior of different polymer structure lightweight meshes used to repair abdominal wall defects.

BACKGROUND: While lightweight (LW) polypropylene (PP) meshes are been used for hernia repair, new prosthetic meshes also of low-density and with large pores have recently been introduced composed of other polymer materials. This study compares the behavior in the short-term of two macroporous LW prosthetic materials, PP and non-expanded PTFE. METHODS: Partial defects were created in the lateral wall of the abdomen in New Zealand White rabbits and then repaired using a LW PP mesh or a new monofile, LW PTFE mesh. At 14 days postimplant, shrinkage and tissue incorporation, gene and protein expression of neo-collagens (qRT-PCR/immunofluorescence), macrophage response (immunohistochemistry) and biomechanical strength were determined. RESULTS: Both meshes induced good host tissue ingrowth, yet the macrophage response was significantly greater for the PTFE implants (p⟨0.05). Collagen 1/3 mRNA expression was greater for the PP mesh but differences lacked significance. Similar patterns of collagen I and III protein expression were observed in the neoformed tissue infiltrating the two meshes. After 14 days of implant, tensile strengths were also similar, while elastic modulus values were higher for the PTFE mesh (p⟨0.05). CONCLUSIONS: In the short term, host collagen deposition and biomechanical performance seemed unaffected by the polymer structure of the implanted mesh. In contrast, the inflammatory response to mesh implant produced at this early time point was more intense for the PTFE.

The angiogenesis promoter, proadrenomedullin N-terminal 20 peptide (PAMP), improves healing in both normoxic and ischemic wounds either alone or in combination with autologous stem/progenitor cells.

A combination of vascular pathologies and other complicating factors results in chronic wounds which constitute a serious burden for both patients and national health systems, due to prolonged hospital stays, high costs, and prolonged nursing staff dedication. Here we investigate whether proadrenomedullin N-terminal 20 peptide (PAMP), a naturally occurring peptide of the skin with antimicrobial and proangiogenic properties, either alone or in combination with autologous skeletal muscle stem/progenitor cells, acts as a wound healing factor. The rabbit ear was chosen as a test system, since it offers a reliable model for normoxic and ischemic wounds. Topical treatments with PAMP, stem/progenitor cells, or a combination of both, resulted in significant improvements of healing, when compared to untreated wounds. PAMP was very effective in promoting reepithelialization and angiogenesis, whereas treatment with stem/progenitor cells alone resulted in less wound contraction. Interestingly, the combination of PAMP and stem/progenitor cells, while maintaining angiogenic potency, reverted to the contraction levels observed in the untreated controls. Under ischemic conditions, generalized necrosis of the dermis and the underlying cartilage was observed in untreated wounds. Treatments of these wounds with PAMP or stem/progenitor cells allowed a timely recovery. In conclusion, PAMP either alone or in combination with autologous stem/progenitor cells may provide a useful tool for improving wound healing both in normoxic and ischemic conditions.

Immune response to the long-term grafting of cryopreserved small-diameter arterial allografts.

INTRODUCTION: The viability and immunological response induced by cryopreserved arterial allografts remain unclear. This study examines the post-graft behaviour of this type of vessel substitute. MATERIALS AND METHODS: Both iliac arteries were extracted from Lewis rats (donors) and used to establish groups of allogeneic fresh (group I) or cryopreserved (group II) grafts in Fisher-344 rats (recipients). Cryopreserved segments for grafting were prepared by automated controlled freezing at a cooling rate of 1°C/min followed by storage in liquid nitrogen vapour at -145°C for 30 days. Before grafting, the vessels were slowly thawed. Animals were sacrificed at 14, 30, 90 and 180 days post-surgery when graft specimens were obtained for light and electron microscopy and immunohistochemical detection of inflammatory cells (CD45, ED1, CD4, CD8). RESULTS: After surgery, 85.71% of the grafts in group I and 82.14% in group II were patent. Following long-term implant, both the fresh and cryopreserved allografts showed complete loss of the muscle compartment of the media. Inflammatory or CD45-positive cells (mainly macrophages and CD8 T-lymphocytes) were detected at earlier time points in suture zones and adventitia. In the fresh allografts, the number of immunolabelled cells steadily increased until they were seen to occupy the entire adventitia at 90 days, with high numbers persisting at 6 months. In the cryopreserved allografts, this adventitial inflammatory infiltrate was significantly reduced. CONCLUSIONS: The cryopreservation/slow thawing protocol used diminished the immune response induced by fresh arterial allografts improving their behaviour after grafting.

Role of lysyl oxidases in neointima development in vascular allografts.

BACKGROUND: Extracellular matrix deposition is the main factor inducing stenotic lesions in arterial grafts. Lysyl oxidases (LOX) play a key role in stabilizing collagen and elastin. OBJECTIVE: To examine the repair response to arterial allografts in terms of LOX expression and collagen/elastin deposition using LOX inhibitors. METHODS: Lewis/Fisher-344 rats were used as donors/recipients. Donor segments were grafted to the right iliac artery of recipients and retrieved 14/30 (short-term) or 90/180 days (long-term) after surgery. One group of animals was injected with a potent irreversible LOX inhibitor daily for 30 days. RESULTS: Intimal hyperplasia increased in thickness until 90/180 days postsurgery. Elastin showed great expression in the neointima at 14/30 days and in the media at 90/180 days. LOX/LOXL1 were similarly expressed in the arterial wall during the first month. In the long term, their overexpression was confined to neointimal layers. At 14 days, collagen types I/III were identified in the grafts. The neointima acquired collagen I over time. In the group of animal treated with the LOX inhibitor, intimal hyperplasia was significantly inhibited. CONCLUSION: LOX were overexpressed in late stages of intimal hyperplasia in the allografts. LOX inhibitors prevented the development of the neointimal layer, such that their modulation could reduce the excessive extracellular matrix deposition that leads to stenosis.

Bioactive polymeric systems with platelet antiaggregating activity for the coating of vascular devices.

The preparation, characterization, and analysis of physicochemical and biological properties of a new bioactive polymer system, based on a copolymer of an acrylic derivative of triflusal (a molecule with chemical structure related to aspirin with antiaggregating activity for platelets) is described and evaluated as thin bioactive coating for vascular grafts and coronary stents. The acrylic monomer derived from triflusal (THEMA) provides random copolymers when it is polymerized with butyl acrylate (BA), according to their reactivity ratios, r(THEMA) = 1.05 and r(BA) = 0.33. The copolymer THBA70, containing a molar composition f(THEMA) = 0.45 and f(BA) = 0.55 presents the optimal properties of stability, flexibility, and adhesion, with a T(g) = 21 ± 2 °C, to be applied as bioactive and biostable coatings for vascular grafts and coronary stents. Thin films of this copolymer system present an excellent biocompatibility and a good inherent antiaggregant activity for platelets.

[Expression of embryonic markers in pterygium derived mesenchymal cells]. / Expresión de marcadores embrionarios en células mesenquimales de pterygium.

BACKGROUND: Destruction of the limbal epithelium barrier is the most important mechanism of pterygium formation (conjunctiva proliferation, encroaching onto the cornea). It is thought to arise from activated and proliferating limbal epithelial stem cells. The objective of this study is to evaluate the presence of undifferentiated mesenchymal cells (stem cells) in cultured cells extracted from human pterygium. MATERIAL AND METHODS: Cells from 6 human pterygium were isolated by explantation and placed in cultures with amniomax medium. Once the monolayer was reached the cells were seeded onto 24 well microplates. The cells were studied in the second sub-culture. The immunohistochemical expression of different embryonic stem cell markers, OCT3/4 and CD9, was analysed. The differentiated phenotypes were characterised with the monoclonal antibodies anti-CD31, α-actin and vimentin. RESULTS: All the cell populations obtained from pterygium showed vimentin expression. Less than 1% of the cells were positive for CD31 and α-actin markers. The majority of the cell population was positive for OCT3/4 and CD9. CONCLUSIONS: The cell population obtained from pterygium expressed mesenchymal cell phenotype and embryonic markers, such us OCT3/4 and CD9. This undifferentiated population could be involved in the large recurrence rate of this type of tissue after surgery.

[Allergic contact dermatitis to diethylthiourea in a neoprene wader]. / Dermatitis alérgica de contacto a dietiltiourea por un traje de pesca de neopreno.

Diethylthiourea, like other thioureas, is often used by the rubber industry and in the manufacture of neoprene. We present a patient who suffered allergic contact dermatitis to diethylthiourea in a neoprene wader and who required admission to hospital and systemic treatment. We review the literature on allergy to diethylthiourea. Thioureas are not included in the standard GEIDAC (Spanish Contact Dermatitis Research Group) battery of patch tests. In these cases, it is necessary to use a special battery of rubber allergens, which includes thiourea compounds, for diagnosis of the disease and to ensure that cases of contact allergic dermatitis to thioureas do not go undiagnosed.

Lysyl oxidase like-1 dysregulation and its contribution to direct inguinal hernia.

OBJECTIVE: The aetiology of inguinal hernia involves changes in collagen turnover and metalloproteinase expression; yet it is not known whether the elastic fibre system could also be affected. This study was designed to compare the expression of tropoelastin (TE), lysyl oxidase-like 1 (LOXL-1) and elastase in the transversalis fascia of patients with and without inguinal hernia. MATERIAL AND METHODS: Transversalis fascia (TF) specimens were obtained from patients undergoing surgery for direct or indirect inguinal hernia (n = 20 each) and from multi-organ donors during organ procurement (controls, n = 16). The specimens were divided according to age (20-40/41-60 years). Tissues were immunohistochemically labelled using anti-tropoelastin, anti-LOXL-1 and anti-elastase antibodies and subjected to Western blot analysis. Relative amounts of LOXL-1 and TE mRNA were determined by real time RT-PCR in cultured cells obtained from the TF of patients and controls. RESULTS: Significantly lower TE and LOXL-1 levels were observed in patients with direct inguinal hernia compared with controls or those with indirect hernia. In contrast, patients with direct inguinal hernia showed significantly higher elastase expression. In fibroblasts isolated from the TF, relative amounts of tropoelastin mRNA were lower for the hernia groups but differences were not significant. LOXL-1 mRNA levels were significantly lower in the direct hernia group compared to controls. CONCLUSIONS: Our findings suggest that impaired elastic fibre function in the transversalis fascia of patients with direct inguinal hernia, reflected by diminished elastin synthesis and its enhanced enzyme degradation, contributes to the development of this type of hernia.

Comparing the behavior of different polypropylene meshes (heavy and lightweight) in an experimental model of ventral hernia repair.

New generation prosthetic biomaterials for abdominal wall repair have been designed to be less dense, by having larger pores than that of the standard polypropylene meshes, to improve abdominal wall compliance. The aim of the present study was to analyze the functional and morphologic properties of these new meshes. For this purpose, 7 x 5 cm(2) defects were created in the anterior abdominal wall of 36 male New Zealand White rabbits and repaired using different polypropylene meshes: a heavyweight mesh (HW), Surgipro, and two lightweight meshes (LW), Parietene and Optilene. Six animals each implanted with biomaterial were sacrificed on postoperative days 14 and 90. Histological and morphometric analysis, adhesion assessment, and biomechanical resistance tests were performed. Similar behavior was shown by the LW and HW meshes in terms of the adhesions and macrophage response induced. After 14 days, the tensile strength of Optilene was greater than the strengths recorded for the other two biomaterials, probably because of its high elasticity. By 90 days, however, the tensile strengths of the three biomaterials were comparable. In conclusion, despite an initial tensile strength advantage shown by the mesh with larger pores, at 90 days postimplant, tensile strengths were similar. Compared with HW, LW prostheses have the benefit that less foreign material was implanted, preserving the elasticity of the recipient host tissue.

Cell viability and prostacyclin release in cultured human umbilical vein endothelial cells.

Construction of efficient substitutes of human blood vessels is strongly dependent on the use of viable and fully functional cultured endothelial cells (ECs). However, very few reports have been published to date focused on the evaluation of cell viability of cultured ECs. In this work, we have determined cell viability, von Willebrand factor, and prostacyclin (PGI(2)) activity in primary cell cultures of human umbilical vein ECs, to identify the specific cell passage that is more appropriate for the development of artificial organs by tissue engineering. Cell viability was determined by quantification of the intracellular concentration of several ions by highly sensitive electron probe X-ray microanalysis, whereas von Willebrand was assayed by immunohistochemistry and PGI(2) release was quantified by radioimmunoassay. The results of our analyses demonstrate that the K/Na ratio was different for each cell passage (4.72 for the first passage, 4.55 for the second passage, and 7.82 for the third passage), suggesting that the highest cell viability corresponds to the third passage. In contrast, PGI(2) production was higher at the first two cell passages, with a significant decrease at the third passage (6.46 +/- 0.10, 5.98 +/- 0.08, and 1.62 +/- 0.05 ng/mL of supernatant for the first, second, and third passages, respectively), whereas von Willebrand expression was similar among the three cell passages analyzed in this work (64.12%, 66.66%, 65.93% of positive cells, respectively). These data suggest that cells corresponding to the second cell passage show the best ratio of viability to functionality and should therefore be used for tissue engineering protocols.