Desregulació proteolítica i inflamatòria tissular en l'hèrnia incisional abdominal humana : paper del fibroblast com a possible factor de risc independent i nova diana terapèutica /

Abdominal incisional hernia (HI) is one of the most common complications of abdominal surgery. The large number of this type of interventions performed annually at hospitals, in addition to high incidence and recurrence rates, are responsible for a significant economic cost to the National Health System. Usually, IH is attributed to technical-related risk factors, such as the type of incision practiced or the applied closure procedure. However, there are sufficient evidences that suggest a biological underlying connective tissue disorder, so-called the biology of hernia, at least in a subset of patients. Recent studies demonstrated the association between collagen diseases and higher IH occurrence rates. Several molecules related to the metabolism of extracellular matrix (ECM) of abdominal tissues have been analyzed, without the molecular complexity achieved in other types of hernia, including groin hernias. However, evidences for patient-related risk factors and also external factors, such as infection, which might seem to favour IH formation suggest that this complication goes beyond a disorder of collagen. The fibroblast is the mostly abundant connective tissue cell and might be mainly involved in the process of IH formation and also in the response to treatment with surgical meshes. The identification of an altered phenotype of fibroblasts from the abdominal tissues may reveal a considerable number of pathways involved in the process of IH formation. The present thesis aims to improve the knowledge of the initiation and formation of IH through identifying cellular and molecular alterations that affect abdominal wall tissues. It also aims to improve the understanding of the cellular response of fibroblast interaction with surgical meshes used for IH treatment in order to identify the routes involved in biointegration. Biopsies from abdominal skeletal muscle and aponeurosis tissues of IH patients were analyzed and compared with those of non-IH patients. The gene expression profiles of skeletal muscle and aponeurosis primary fibroblasts extracted from both patients were also compared. Finally, the behaviour of the aponeurosis fibroblasts from both patients in contact with polypropylene meshes used for IH treatment were compared. A dysfunction in abdominal tissues of IH patients, compared with non-IH patients, in addition to some alterations in the ECM metabolism associated with inflammatory molecular signalling deregulation has been observed. Specifically, an altered MMPs/TIMPs balance in favour of net proteolytic activity and a deregulated signalling and levels of certain pro-inflammatory cytokines has been found. Although these changes are tissue-specific lead to similar consequences. In addition, a differential gene expression profile using DNA microarrays has been identified in fibroblasts from IH abdominal tissues. It has been observed that the altered pathways obtained in fibroblasts from aponeurosis are related to cell cycle and cellular damage repair while in fibroblasts from skeletal muscle are involved in the immune response. In addition, a set of genes that may have a significant relevance in the process of IH formation have been identified. Finally, an in vitro model for studying the interaction between aponeurosis primary fibroblasts and polypropylene meshes has been established. It has been observed that cell adhesion to meshes is weak and could lead to changes in cytoskeletal molecules organization that could lead to cell death. In addition, the polypropylene mesh may modify the altered cell cycle of fibroblasts form IH patients mitigating their phenotype.L'hèrnia incisional (HI) abdominal és una de les complicacions més freqüents secundàries a una intervenció quirúrgica abdominal. L'elevat nombre d'intervencions d'aquest tipus que es realitzen anualment als hospitals, a més de les taxes elevades d'incidència i recurrència, són responsables que aquest desordre generi una despesa econòmica important per al Sistema Nacional de Salut. Generalment, l'aparició de l'HI s'atribueix a factors tècnics durant l'operació, com el tipus d'incisió practicada o el tipus de tancament aplicat. De tota manera, hi ha prou evidències per considerar que, almenys en un subgrup de pacients, podria haver-hi una alteració del teixit connectiu subjacent que permetria parlar d'una biologia de l'hèrnia. Estudis recents han evidenciat l'associació entre malalties associades amb alteracions en el col·lagen i una major probabilitat d'aparició de l'HI. En aquest sentit, s'ha analitzat algunes molècules relacionades amb el metabolisme de la matriu extracel·lular (MEC) dels teixits abdominals però sense arribar a la profunditat molecular d'altres tipus d'hèrnies, com la inguinal. De tota manera, l'evidència de factors de risc inherents al pacient o externs, com la infecció, sembla que podrien afavorir la formació de l'HI i posen de manifest que el problema va més enllà d'un desordre en el col·lagen. El fibroblast, com a cèl·lula predominant del teixit connectiu, podria ser la cèl·lula majoritàriament implicada en el procés i en la resposta al tractament mitjançant malles quirúrgiques. La identificació del fenotip alterat dels fibroblasts procedents dels teixits abdominals podria posar al descobert una sèrie de rutes implicades en el procés de formació de l'HI. En la present tesi es pretén millorar el coneixement del procés d'iniciació i formació de l'HI a través de la identificació de les alteracions al nivell cel·lular i molecular que afecten els teixits que conformen la paret abdominal. A més, també es pretén millorar el coneixement de la resposta cel·lular al tractament amb malles quirúrgiques per identificar les rutes implicades en la biointegració. Es van analitzar biòpsies de múscul esquelètic i d'aponeurosi de la zona abdominal de pacients amb HI i es van comparar amb els de pacients sense HI. També es va comparar els perfils d'expressió gènica dels fibroblasts primaris procedents d'ambdós grups de pacients. Finalment, es va comparar el comportament dels fibroblasts procedents d'aponeurosi d'ambdós grups de pacients en contacte amb malles de polipropilè utilitzades per al tractament de l'HI. S'ha observat una desestructuració dels teixits abdominals dels pacients amb HI, en comparació amb els de pacients sense HI, a més d'unes alteracions en el metabolisme de la MEC associades a una desregulació en la senyalització molecular inflamatòria. Concretament, s'ha observat una relació MMPs/TIMPs alterada en favor d'una major activitat proteolítica i una desregulació dels nivells i la regulació d'algunes citocines pro-inflamatòries. Aquests alteracions són teixit-específiques però desemboquen en conseqüències similars. A més, s'ha identificat un perfil d'expressió gènica diferencial mitjançant l'ús de xips d'expressió gènica en els fibroblasts procedents dels teixits abdominals. S'ha observat que les rutes alterades en els fibroblasts procedents d'aponeurosi guarden relació amb el cicle cel·lular i la reparació del dany cel·lular, mentre que en el múscul esquelètic participen en la resposta immunològica. A més, s'han identificat un conjunt de gens que poden tenir una importància destacada en el procés de formació de l'HI. Finalment, s'ha establert un model d'estudi in vitro de la interacció dels fibroblasts primaris procedents d'aponeurosi amb malles quirúrgiques de polipropilè. S'ha observat que l'adhesió cel·lular és feble i que podria produir canvis en l'organització de molècules del citoesquelet que podrien desembocar en la mort cel·lular. A més, la malla de polipropilè podria modificar el cicle cel·lular alterat dels fibroblasts de pacients amb HI atenuant el seu fenotip

The influence of physicochemical properties of biomimetic Hydroxyapatite on the in vitro behavior of endothelial progenitor cells and their interaction with mesenchymal stem cells

Calcium phosphate (CaP) substrates are successfully used as bone grafts due to their osteogenic properties. However, the influence of the physicochemical features of CaPs in angiogenesis is frequently neglected despite it being a crucial process for bone regeneration. The present work focuses on analyzing the effects of textural parameters of biomimetic calcium deficient hydroxyapatite (CDHA) and sintered beta-tricalcium phosphate (ß-TCP), such as specific surface area, surface roughness, and microstructure, on the behavior of rat endothelial progenitor cells (rEPCs) and their crosstalk with rat mesenchymal stem cells (rMSCs). The higher reactivity of CDHA results in low proliferation rates in monocultured and cocultured systems. This effect is especially pronounced for rMSCs alone, and for CDHA with a fine microstructure. In terms of angiogenic and osteogenic gene expressions, the upregulation of particular genes is especially enhanced for needle-like CDHA compared to plate-like CDHA and ß-TCP, suggesting the importance not only of the chemistry of the substrate, but also of its textural features. Moreover, the coculture of rEPCs and rMSCs on needle-like CDHA results in early upregulation of osteogenic modulator, i.e., protein deglycase 1 might be a possible cause of overexpression of osteogenic-related genes on the same substrate.Peer ReviewedPostprint (published version

Com es forma una hèrnia incisional?

Una tesi llegida a la UAB ha buscat millorar el coneixement del procés d'iniciació i formació de l'hèrnia incisional (HI) abdominal, una de les complicacions més freqüents que apareixen posteriorment a una intervenció quirúrgica abdominal de qualsevol tipus. Aquest treball es va realitzar mitjançant la identificació de les alteracions al nivell cel·lular i molecular que afecten els teixits que conformen la paret abdominal. Aquest és un desordre que genera una despesa econòmica important, ja que es dona en un gran nombre de pacients després d'intervencions quirúrgiques abdominals.Una tesis leída en la UAB ha buscado mejorar el conocimiento del proceso de iniciación y formación de la hernia incisional (HI) abdominal, una de las complicaciones más frecuentes que aparecen posteriormente a una intervención quirúrgica abdominal de cualquier tipo. Este trabajo se realizó mediante la identificación de las alteraciones en el nivel celular y molecular que afectan a los tejidos que conforman la pared abdominal. Este es un desorden que genera un gasto económico importante, ya que se dan un gran número de casos después de intervenciones quirúrgicas abdominales

Guiding fibroblast activation using an RGD-mutated heparin binding II fragment of fibronectin for gingival titanium integration

The formation of a biological seal around the neck of titanium (Ti) implants is critical for ensuring integration at the gingival site and for preventing bacterialcolonization that may lead to periimplantitis. This process is guided byactivated fibroblasts, named myofibroblasts, which secrete extracellularmatrix (ECM) proteins and ECM-degrading enzymes resolving the wound.However, in some cases, Ti is not able to attract and activate fibroblasts to asufficient extent, which may compromise the success of the implant.Fibronectin (FN) is an ECM component found in wounds that is able to guidesoft tissue healing through the adhesion of cells and attraction of growthfactors (GFs). However, clinical use of FN functionalized Ti implants isproblematic because FN is difficult to obtain, and is sensitive to degradation.Herein, functionalizing Ti with a modified recombinant heparin binding II(HBII) domain of FN, mutated to include an Arg-Gly-Asp (RGD) sequence forpromoting both fibroblast adhesion and GF attraction, is aimed at. TheHBII-RGD domain is able to stimulate fibroblast adhesion, spreading,proliferation, migration, and activation to a greater extent than the nativeHBII, reaching values closer to those of full-length FN suggesting that itmight induce the formation of a biological sealing.Peer ReviewedPostprint (published version

Reducing bacterial adhesion to titanium surfaces using low intensity alternating electrical pulses

BACKGROUND Orthopedic implant-related infection remains one of the most serious complications after orthopedic surgery. In recent years, there has been an increased scientific interest to improve prevention and treatment strategies. However, many of these strategies have focused on chemical measures. AIM To analyze the effect of alternating current electrical fields on bacterial adherence to titanium surfaces. METHODS Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were exposed to 6.5 V electrical currents at different frequencies: 0.5 Hz, 0.1 Hz, and 0.05 Hz. After exposure, a bacterial count was then performed and compared to the control model. Other variables registered included the presence of electrocoagulation of the medium, electrode oxidation and/or corrosion, and changes in pH of the medium. RESULTS The most effective electrical model for reducing S. aureus adhesion was 6.5 V alternating current at 0.05 Hz achieving a 90% adhesion reduction rate. For E. coli, the 0.05 Hz frequency model also showed the most effective results with a 53% adhesion reduction rate, although these were significantly lower than S. aureus. Notable adhesion reduction rates were observed for S. aureus and E.coli in the studied conditions. However, the presence of electrode oxidation makes us presume these conditions are not optimal for in vivo use. CONCLUSION Although our findings suggest electrical currents may be useful in preventing bacterial adhesion to metal surfaces, further research using other electrical conditions must be examined to consider their use for in vivo trials.Peer ReviewedPostprint (published version

Injectable calcium phosphate foams for the delivery of Pitavastatin as osteogenic and angiogenic agent

Apatitic bone cements have been used as a clinical bone substitutes and drug delivery vehicles for therapeutic agents in orthopedic applications. This has led to their combination with different drugs with known ability to foster bone formation. Recent studies have evaluated Simvastatin for its role in enhanced bone regeneration, but its lipophilicity hampers incorporation and release to and from the bone graft. In this study, injectable calcium phosphate foams (i-CPF) based on a-tricalcium phosphate were loaded for the first time with Pitavastatin. The stability of the drug in different conditions relevant to this study, the effect of the drug on the i-CPFs properties, the release profile, and the in vitro biological performance with regard to mineralization and vascularization were investigated. Pitavastatin did not cause any changes in neither the micro nor the macro structure of the i-CPFs, which retained their biomimetic features. PITA-loaded i-CPFs showed a dose-dependent drug release, with early stage release kinetics clearly affected by the evolving microstructure due to the setting of cement. in vitro studies showed dose-dependent enhancement of mineralization and vascularization. Our findings contribute towards the design of controlled release with low drug dosing bone grafts: i-CPFs loaded with PITA as osteogenic and angiogenic agentPeer ReviewedPostprint (author's final draft

Antimicrobial PHAs coatings for solid and porous tantalum implants

Biomaterial-associated infections (BAI) are the major cause of failure of indwelling medical devices. The risk of BAI can end dramatically in the surgical removal of the affected device. Therefore, a major effort must be undertaken to guarantee the permanence of the implant. In this regard, we have developed antimicrobial coatings for tantalum (Ta) implants, using polyhydroxyalkanoates (PHAs) as matrices for carrying an active principle. The dip-coating technique was successfully used for covering solid Ta discs. An original PHA emulsion flow process was developed for the coating of porous Ta structures, specially for the inner surfaces. The complete characterization of the biopolymer coatings, their antibacterial properties, toxicity and biointegration were analyzed. Thus, non-toxic, well-biointegrated homogeneous biopolymer coatings were attained, which showed antibacterial properties. By using biodegradable PHAs, the resulting drug delivery system assured the protection of Ta against bacterial infections for a period of time.Peer ReviewedPostprint (author's final draft

Mechanical and microstructural characterization of new nickel-free low modulus beta-type titanium wires during thermomechanical treatments

NiTi alloy is the only practical shape memory alloy (SMA) in biomedical use because of its excellent mechanical stability and functionality. However, it is estimated that between 4.5% and 28.5% of the population are hypersensitive to nickel metal, with a higher prevalence in females. Therefore, developing nickel-free low modulus beta-type titanium alloys showing shape memory or super elastic behavior would have a great interest in the biomaterials field. Homogeneous 127 mu m diameter Ti25Hf21Nb wires were produced and compared to straight annealed Ti-50.8 at% Ni (Nitinol) and 90% cold-drawn 316L wires. Microstructural changes taking place during the heat treatment of cold-worked Ti25Hf21Nb wires were investigated. Large plastic deformation during wire drawing and subsequent annealing led to nano-crystallization and amorphization which may contribute to the observed superelasticity. Mechanical properties were characterized using cyclic uniaxial tension and rotary beam fatigue test modes. A modulus of elasticity of less than 60 GPa and axial recoverable strain of greater than 3% were observed with stress hysteresis resembling a reversible stress-induced martensitic transformation at higher temperatures. The new Ti25Hf21Nb alloy is an important candidate for developing Ni-free SMAs in the future. (C) 2015 Elsevier B.V. All rights reserved.Peer ReviewedPostprint (author's final draft

Effect of nano-structural properties of biomimetic hydroxyapatite on osteoimmunomodulation

Immune cells are sensitive to the microstructural and textural properties of materials. Tuning the structural features of synthetic bone grafts could be a valuable strategy to regulate the specific response of the immune system, which in turn modulates the activity of bone cells. The aim of this study was to analyse the effect of the structural characteristics of biomimetic calcium deficient hydroxyapatite (CDHA) on the innate immune response of macrophages and the subsequent impact on osteogenesis and osteoclastogenesis. Murine RAW 264.7¿cells were cultured, under standard and inflammatory conditions, on chemically identical CDHA substrates that varied in microstructure and porosity. The impact on osteogenesis was evaluated by incubating osteoblastic cells (SaOS-2) with RAW-CDHA conditioned extracts. The results showed that macrophages were sensitive to different textural and structural properties of CDHA. Under standard conditions, the impact of inflammatory cytokine production by RAW cells cultured on CDHA played a significant role in the degradation of substrates, suggesting the impact of resorptive behaviour of RAW cells on biomimetic surfaces. Osteoblast differentiation was stimulated by the conditioned media collected from RAW cells cultured on needle-like nanostructured CDHA. The results demonstrated that needle-like nanostructured CDHA was able to generate a favourable osteoimmune environment to regulate osteoblast differentiation and osteogenesis. Under inflammatory conditions, the incubation of RAW cells with less porous CDHA resulted in a decreased gene expression and release of pro-inflammatory cytokines.Peer ReviewedPostprint (author's final draft

Plasma-induced selectivity in bone cancer cells death

Background Current therapies for bone cancers - either primary or metastatic – are difficult to implement and unfortunately not completely effective. An alternative therapy could be found in cold plasmas generated at atmospheric pressure which have already demonstrated selective anti-tumor action in a number of carcinomas and in more relatively rare brain tumors. However, its effects on bone cancer are still unknown. Methods Herein, we employed an atmospheric pressure plasma jet (APPJ) to validate its selectivity towards osteosarcoma cell line vs. osteoblasts & human mesenchymal stem cells. Results Cytotoxicity following direct interaction of APPJ with cells is comparable to indirect interaction when only liquid medium is treated and subsequently added to the cells, especially on the long-term (72 h of cell culture). Moreover, following contact of the APPJ treated medium with cells, delayed effects are observed which lead to 100% bone cancer cell death through apoptosis (decreased cell viability with incubation time in contact with APPJ treated medium from 24 h to 72 h), while healthy cells remain fully viable and unaffected by the treatment. Conclusions The high efficiency of the indirect treatment indicates that an important role is played by the reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the gaseous plasma stage and then transmitted to the liquid phase, which overall lead to lethal and selective action towards osteosarcoma cells. These findings open new pathways for treatment of metastatic bone disease with a minimally invasive approach.Peer ReviewedPostprint (author's final draft