Microrheological comparison of melanoma cells by atomic force microscopy.

Melanoma is one of the most severe cancers due to its great potential to form metastasis. Recent studies showed the importance of mechanical property assessment in metastasis formation which depends on the cytoskeleton dynamics and cell migration. Although cells are considered purely elastic, they are viscoelastic entities. Microrheology atomic force microscopy (AFM) enables the assessment of elasticity and viscous properties, which are relevant to cell behavior regulation. The current work compares the mechanical properties of human neonatal primary melanocytes (HNPMs) with two melanoma cell lines (WM793B and 1205LU cells), using microrheology AFM. Immunocytochemistry of F-actin filaments and phosphorylated focal adhesion kinase (p-FAK) and cell migration assays were performed to understand the differences found in microrheology AFM regarding the tumor cell lines tested. AFM revealed that HNPMs and tumor cell lines had distinct mechanical properties. HNPMs were softer, less viscous, presenting a higher power-law than melanoma cells. Immunostaining showed that metastatic 1205LU cells expressed more p-FAK than WM793B cells. Melanoma cell migration assays showed that WM73B did not close the gap, in contrast to 1205LU cells, which closed the gap at the end of 23 h. These data seem to corroborate the high migratory behavior of 1205LU cells. Microrheology AFM applied to HNPMs and melanoma cells allowed the quantification of elasticity, viscous properties, glassy phase, and power-law properties, which have an impact in cell migration and metastasis formation. AFM study is important since it can be used as a biomarker of the different stages of the disease in melanoma.

New prospects in skin regeneration and repair using nanophased hydroxyapatite embedded in collagen nanofibers.

This study reflects an exploitation of a composite matrix produced by electrospinning of collagen and electrospraying of nanophased hydroxyapatite (nanoHA), for skin regeneration applications. The main goal was to evaluate the effect of nanoHA, as source of localized calcium delivery, on human dermal fibroblasts, keratinocytes, and human mesenchymal stem cells (hMSCs) growth, proliferation, differentiation, and extracellular matrix production. This study revealed that calcium ions provided by nanoHA significantly enhanced cellular growth and proliferation rates and prevented adhesion of pathogenic bacteria strains typically found in human skin flora. Moreover, hMSCs were able to differentiate in both osteogenic and adipogenic lineages. Rat subcutaneous implantation of the membranes also revealed that no adverse reaction occurred. Therefore, the mechanically fit composite membrane presents a great potential to be used either as cell transplantation scaffold for skin wound regeneration or as wound dressing material in plastic surgery, burns treatment or skin diseases.

Role of SPARC in bone remodeling and cancer-related bone metastasis.

There is a growing socioeconomic recognition that clinical bone diseases such as bone infections, bone tumors and osteoporotic bone loss mainly associated with ageing, are major issues in today's society. SPARC (secreted protein, acidic and rich in cysteine), a matricellular glycoprotein, may be a promising therapeutic target for preventing or treating bone-related diseases. In fact, SPARC is associated with tissue remodeling, repair, development, cell turnover, bone mineralization and may also participate in growth and progression of tumors, namely cancer-related bone metastasis. Yet, the function of SPARC in such biological processes is poorly understood and controversial. The main objective of this work is to review the current knowledge related to the activity of SPARC in bone remodeling, tumorigenesis, and bone metastasis. Progress in understanding SPARC biology may provide novel strategies for bone regeneration and the development of anti-angiogenic, anti-proliferative, or counter-adhesive treatments specifically against bone metastasis.

Local Antibiotic Delivery Ceramic Bone Substitutes for the Treatment of Infected Bone Cavities and Bone Regeneration: A Systematic Review on What We Have Learned from Animal Models.

AIMS: the focus of this study is to evaluate if the combination of an antibiotic with a ceramic biomaterial is effective in treating osteomyelitis in an infected animal model and to define which model and protocol are best suited for in vivo experiments of local bone infection treatment. METHODS: a systematic review was carried out based on PRISMA statement guidelines. A PubMed search was conducted to find original papers on animal models of bone infections using local antibiotic delivery systems with the characteristics of bone substitutes. Articles without a control group, differing from the experimental group only by the addition of antibiotics to the bone substitute, were excluded. RESULTS: a total of 1185 records were retrieved, and after a three-step selection, 34 papers were included. Six manuscripts studied the effect of antibiotic-loaded biomaterials on bone infection prevention. Five articles studied infection in the presence of foreign bodies. In all but one, the combination of an antibiotic with bioceramic bone substitutes tended to prevent or cure bone infection while promoting biomaterial osteointegration. CONCLUSIONS: this systematic review shows that the combination of antibiotics with bioceramic bone substitutes may be appropriate to treat bone infection when applied locally. The variability of the animal models, time to develop an infection, antibiotic used, way of carrying and releasing antibiotics, type of ceramic material, and endpoints limits the conclusions on the ideal therapy, enhancing the need for consistent models and guidelines to develop an adequate combination of material and antimicrobial agent leading to an effective human application.

Studying the Degradation of Three Polymers under Different Chlorine Concentrations and Exposure Times.

Due to chlorine's ability to kill bacteria and fungi through a chemical reaction, chlorine solutions are commonly used to clean and disinfect numerous public facilities, although these actions are also dependent to the equipment present in those facilities. Accordingly, the interest in studying its effect when in contact with different materials is obvious. This study was carried out through accelerated degradation tests and various analysis methods (optical microscope, scanning electron microscope, and tensile tests). The objective was to observe the wear presented by three polymeric materials, polyvinyl chloride (PVC), high-density polyethylene (HDPE), and polypropylene (PP), when exposed to chlorine's action in swimming pools and drinking water treatment plants. The resulting effect depends on the chlorine content and the type of contact between the chemical agent and the material. The aim was to select the material less likely to be affected by chlorine through tests and analyses, allowing a longer component life. The use of certain more resistant polymeric materials can drastically reduce maintenance, reducing fundamental factors such as costs, the downtime of municipal facilities, and also the risk to public health. It was concluded that PVC has the most stable behaviour overall when in contact with chlorine solutions.

Study of the Chlorine Influence on the Corrosion of Three Steels to Be Used in Water Treatment Municipal Facilities.

Many municipal facilities, such as pools and drinking water treatment facilities, are subject to ongoing maintenance due to the corrosion of their metallic materials caused by chlorine, leading to high costs and a possible risk to public health. A proper study of the employed product's effect could lead to the use of better materials, which significantly increase the lifetime of metallic equipment more attacked by corrosion, through studies evaluating their cost-effectiveness. This paper was carried out with the objective of studying the degradation of some metallic materials (AISI 316L, AISI 321 and Duplex 14462) used in the referred facilities in order to select the one that possessed a better behavior. It was observed that the introduction of some more adequate materials can drastically reduce maintenance operations, with Duplex 14462 showing the best results, ideal for greater chlorine concentrations, followed by AISI 321, which may be employed for components in less contact with chlorine, since it is more easily affordable.

The Impact of the Deterioration on Wood by Chlorine: An Experimental Study.

The use of disinfection and cleaning chemicals in several municipal facilities, such as swimming pools and drinking water treatment plants, causes the degradation of various types of wood, which leads to failures in equipment and the corresponding need for maintenance. This degradation creates added costs for municipalities, as well as the closure of certain facilities due to curative or preventive maintenance and, in many cases, public health issues, due to the water being contaminated with deteriorating products. Through a thorough study of the degradation effect on the products, more resistant materials can be found which are able to withstand these adversities and increase the lifespan of wood in regular contact with chemical agents. This is achievable by the determination of the cost-effectiveness of the substitute material to replace these components with alternative ones, with properties that better resist the deterioration effects promoted by aggressive environments. No studies have been found so far strictly focused on this matter. The objective of this study is to evaluate the degradation presented by two types of wood, beech and oak, which are exposed to the action of chlorine in municipal facilities. This degradation varies according to the chlorine content and the materials' time of contact with the chemical agent, allowing the selection of new materials which will provide an extended lifetime of the components, reducing maintenance drastically, as well as costs for the facilities and the risk to public health. The performed experimental tests have shown that the oak wood has the best results regarding chlorine degradation resistance.

Vancomycin-Loaded, Nanohydroxyapatite-Based Scaffold for Osteomyelitis Treatment: In Vivo Rabbit Toxicological Tests and In Vivo Efficacy Tests in a Sheep Model.

The treatment for osteomyelitis consists of surgical debridement, filling of the dead space, soft tissue coverage, and intravenous administration of antimicrobial (AM) agents for long periods. Biomaterials for local delivery of AM agents, while providing controllable antibiotic release rates and simultaneously acting as a bone scaffold, may be a valuable alternative; thus, avoiding systemic AM side effects. V-HEPHAPC is a heparinized nanohydroxyapatite (nHA)/collagen biocomposite loaded with vancomycin that has been previously studied and tested in vitro. It enables a vancomycin-releasing profile with an intense initial burst, followed by a sustained release with concentrations above the Minimum Inhibitory Concentration (MIC) for MRSA. In vitro results have also shown that cellular viability is not compromised, suggesting that V-HEPHAPC granules may be a promising alternative device for the treatment of osteomyelitis. In the present study, V-HEPHAPC (HEPHAPC with vancomycin) granules were used as a vancomycin carrier to treat MRSA osteomyelitis. First, in vivo Good Laboratory Practice (GLP) toxicological tests were performed in a rabbit model, assuring that HEPHAPC and V-HEPHAPC have no relevant side effects. Second, V-HEPHAPC proved to be an efficient drug carrier and bone substitute to control MRSA infection and simultaneously reconstruct the bone cavity in a sheep model.

Mechanobiology of Colorectal Cancer.

In this review, the mechanobiology of colorectal cancer (CRC) are discussed. Mechanotransduction of CRC is addressed considering the relationship of several biophysical cues and biochemical pathways. Mechanobiology is focused on considering how it may influence epithelial cells in terms of motility, morphometric changes, intravasation, circulation, extravasation, and metastization in CRC development. The roles of the tumor microenvironment, ECM, and stroma are also discussed, taking into account the influence of alterations and surface modifications on mechanical properties and their impact on epithelial cells and CRC progression. The role of cancer-associated fibroblasts and the impact of flow shear stress is addressed in terms of how it affects CRC metastization. Finally, some insights concerning how the knowledge of biophysical mechanisms may contribute to the development of new therapeutic strategies and targeting molecules and how mechanical changes of the microenvironment play a role in CRC disease are presented.

Mechanical Properties of Colorectal Cancer Cells Determined by Dynamic Atomic Force Microscopy: A Novel Biomarker.

Colorectal cancer (CRC) has been addressed in the framework of molecular, cellular biology, and biochemical traits. A new approach to studying CRC is focused on the relationship between biochemical pathways and biophysical cues, which may contribute to disease understanding and therapy development. Herein, we investigated the mechanical properties of CRC cells, namely, HCT116, HCT15, and SW620, using static and dynamic methodologies by atomic force microscopy (AFM). The static method quantifies Young's modulus; the dynamic method allows the determination of elasticity, viscosity, and fluidity. AFM results were correlated with confocal laser scanning microscopy and cell migration assay data. The SW620 metastatic cells presented the highest Young's and storage moduli, with a defined cortical actin ring with distributed F-actin filaments, scarce vinculin expression, abundant total focal adhesions (FAK), and no filopodia formation, which could explain the lessened migratory behavior. In contrast, HCT15 cells presented lower Young's and storage moduli, high cortical tubulin, less cortical F-actin and less FAK, and more filopodia formation, probably explaining the higher migratory behavior. HCT116 cells presented Young's and storage moduli values in between the other cell lines, high cortical F-actin expression, intermediate levels of total FAK, and abundant filopodia formation, possibly explaining the highest migratory behavior.

Combining local antibiotic delivery with heparinized nanohydroxyapatite/collagen bone substitute: A novel strategy for osteomyelitis treatment.

Osteomyelitis is a major challenge in bone surgery and conventional treatment is frequently ineffective to control the infection, with an alternative approach being required. In the present work, a heparinized nanohydroxyapatite/collagen biocomposite was produced in granular form, and loaded with vancomycin, to work as a local drug delivery system for osteomyelitis and as a bone substitute. This strategy involves the local delivery of high concentrations of vancomycin, to eradicate the infection. Additionally, these granules work as a scaffold with regenerative properties, to induce bone regeneration after antibiotic release. The heparinized nanohydroxyapatite/collagen granular bone substitute was produced using two different sintering temperatures to study their effect on granules properties and on vancomycin release profile. Morphological, topographic, chemical and mechanical characterization were carried out for granules sintered at both temperatures and some relevant differences were found. The mechanical strength was increased by several orders of magnitude with increasing sintering temperature, being able to maintain their porous macrostructure and withstand important processes for their commercialization such as packaging, shipping and surgical manipulation. The nanohydroxyapatite/collagen granules were able to release high concentrations of vancomycin, always above MIC, for 19 days. The released antibiotic was able to eradicate both planktonic and sessile methicillin-resistant Staphylococcus aureus. The cytotoxicity was assessed according to ISO 10993-5:2009 and the granules sintered at higher temperature showed no cytotoxic effect. Considering these results nanohydroxyapatite/collagen biocomposite loaded with vancomycin is a promising solution for osteomyelitis treatment.

Melanoma in the Eyes of Mechanobiology.

Skin is the largest organ of the human body with several important functions that can be impaired by injury, genetic or chronic diseases. Among all skin diseases, melanoma is one of the most severe, which can lead to death, due to metastization. Mechanotransduction has a crucial role for motility, invasion, adhesion and metastization processes, since it deals with the response of cells to physical forces. Signaling pathways are important to understand how physical cues produced or mediated by the Extracellular Matrix (ECM), affect healthy and tumor cells. During these processes, several molecules in the nucleus and cytoplasm are activated. Melanocytes, keratinocytes, fibroblasts and the ECM, play a crucial role in melanoma formation. This manuscript will address the synergy among melanocytes, keratinocytes, fibroblasts cells and the ECM considering their mechanical contribution and relevance in this disease. Mechanical properties of melanoma cells can also be influenced by pigmentation, which can be associated with changes in stiffness. Mechanical changes can be related with the adhesion, migration, or invasiveness potential of melanoma cells promoting a high metastization capacity of this cancer. Mechanosensing, mechanotransduction, and mechanoresponse will be highlighted with respect to the motility, invasion, adhesion and metastization in melanoma cancer.

The antibacterial and angiogenic effect of magnesium oxide in a hydroxyapatite bone substitute.

Bone graft infections are serious complications in orthopaedics and the growing resistance to antibiotics is increasing the need for antibacterial strategies. The use of magnesium oxide (MgO) is an interesting alternative since it possesses broad-spectrum antibacterial activity. Additionally, magnesium ions also play a role in bone regeneration, which makes MgO more appealing than other metal oxides. Therefore, a bone substitute composed of hydroxyapatite and MgO (HAp/MgO) spherical granules was developed using different sintering heat-treatment cycles to optimize its features. Depending on the sintering temperature, HAp/MgO spherical granules exhibited distinct surface topographies, mechanical strength and degradation profiles, that influenced the in vitro antibacterial activity and cytocompatibility. A proper balance between antibacterial activity and cytocompatibility was achieved with HAp/MgO spherical granules sintered at 1100 ºC. The presence of MgO in these granules was able to significantly reduce bacterial proliferation and simultaneously provide a suitable environment for osteoblasts growth. The angiogenic and inflammation potentials were also assessed using the in vivo chicken embryo chorioallantoic membrane (CAM) model and the spherical granules containing MgO stimulated angiogenesis without increasing inflammation. The outcomes of this study evidence a dual effect of MgO for bone regenerative applications making this material a promising antibacterial bone substitute.

Antibacterial bone substitute of hydroxyapatite and magnesium oxide to prevent dental and orthopaedic infections.

Bone substitutes market is growing due to the great demand for bone regenerative therapies. However, most of the actual products available in the market are incapable of inhibiting bacterial colonization, which can lead to tissue infection and possible implant failure. Some bone substitutes are combined with antibiotics to avoid the development of implant-associated infections, but the growing bacterial resistance to antibiotics often makes these products ineffective. Therefore, it is mandatory to develop new and alternative approaches. In the present work, a granular bone substitute of hydroxyapatite was produced, where different percentages of magnesium oxide were introduced. The antibacterial activity and biofilm formation was evaluated towards Staphylococcus aureus and Escherichia coli. The inclusion of magnesium oxide particles reduced bacterial growth and biofilm formation in a concentration-dependent manner, when compared with pure hydroxyapatite materials. Superior antibacterial activity and inhibition of biofilm formation was observed for Staphylococcus aureus with complete eradication when magnesium oxide percentages were equal or above 3 wt%. The materials cytotoxicity was assessed under ISO 10993-5:2009 guidance and through Live/Dead cell marking and none of the produced granules was cytotoxic. In addition, reactive oxygen species production was also evaluated and the results revealed that the exposure to the materials extracts did not induce the formation of reactive oxygen species by cells.

Influence of PLLA/PCL/HA Scaffold Fiber Orientation on Mechanical Properties and Osteoblast Behavior.

Scaffolds based on aligned and non-aligned poly (L-lactic acid) (PLLA)/polycaprolactone (PCL) fibers obtained by electrospinning, associated to electrosprayed hydroxyapatite (HA) for tissue engineering applications were developed and their performance was compared in terms of their morphology and biological and mechanical behaviors. The morphological results assessed by scanning electron microscopy showed a mesh of PLLA/PCL fibers (random and perfectly aligned) associated with aggregates of nanophased HA. Fourier transform infrared spectrometry confirmed the homogeneity in the blends and the presence of nanoHA in the scaffold. As a result of fiber alignment a 15-fold increase in Young's Modulus and an 8-fold increase in tensile strength were observed when compared to non-aligned fibers. In PLLA/PCL/HA scaffolds, the introduction of nanoHA caused a remarkable improvement of the mechanical strength of this material acting as a reinforcement, enhancing the response of these constructs to tensile stress. In vitro testing was evaluated using osteoblast (MC3T3-E1) cells. The results showed that both fibrous scaffolds were able to support osteoblast cell adhesion and proliferation and that fiber alignment induced increased cellular metabolic activity. In addition, the adhesion and proliferation of Staphylococcus aureus were evaluated and a lower number of colony forming units (CFUs) was obtained in the scaffolds with aligned fibers.

Individual and mixture toxicity evaluation of three pharmaceuticals to the germination and growth of Lactuca sativa seeds.

This work aims to assess, individually and in mixtures, possible phytotoxic effects of three pharmaceuticals (paracetamol, ibuprofen and amoxicillin) on germination and early growth of Lactuca sativa seeds. Pharmaceuticals are an important group of emerging contaminants, whose presence has been described in several environmental compartments, including soils. However, knowledge on their possible impact in terrestrial organisms is still sparse and even more when mixtures are considered. Germination tests are important to evaluate the quality of soil and the toxic effects that contaminants can pose to plants. The acute effects of individual pharmaceuticals as well as binary and ternary mixtures were assessed using different endpoints, namely: percentage of seed germination, root elongation, shoot and leaf length, after an exposure time of five days. Overall, in the exposure of L. sativa seeds to individual pharmaceuticals there are indications of acute toxicity in the early plant growth. However, this inhibitory effect tends to be cancelled in the acute exposure to mixtures. This study shows the importance of evaluating the toxicity of mixtures of pharmaceuticals, since they might have distinct toxic effects when compared to the single compounds, and also because, probably, it is the closest scenario to the reality that can be found in the environment.

Behavior of prostate cancer cells in a nanohydroxyapatite/collagen bone scaffold.

Prostate cancer (PCa) is the second leading cause of death among men in Europe and U.S. The metastatic dissemination pattern of PCa is unique, developing bone metastasis as the only site of progression, consequently with a prognosis very poor. The cancer cells interactions within the surrounding bone environment are critical for tumor growth and progression. Secreted protein, acidic and rich in cysteine (SPARC) is described to be involved in PCa cells migration and invasion into bone. Three-dimensional (3D) in vitro systems that are able to closely resemble the in vivo microenvironment are recently taking importance in cancer research. Original nanohydroxyapatite/collagen scaffolds were designed to resemble bone microenvironment in order to be applied as substitutes in bone defects and as potential biomaterials to mimic skeletal tumors. In fact, these 3D structures were cytocompatible and able to support osteoblast (MC3T3-E1) colonization and to promote bone ingrowth. Additionally, SPARC adsorption onto the scaffolds affected PC3 and LNCaP PCa cell lines behavior. PC3 cells were found to adapt and colonize the scaffolds, differing from LNCaP where cells underwent morphogenic changes and grew as clusters. Furthermore, for the tested SPARC concentration, SPARC plays a role in retaining LNCaP cells at the latter time points while with PC3 cells no significant differences were observed. This characterization study is required to establish a bone model to provide new insights into the poorly understood PCa mechanisms of metastasis to bone and the generation of improved therapies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2035-2046, 2017.

Injectable hybrid system for strontium local delivery promotes bone regeneration in a rat critical-sized defect model.

Strontium (Sr) has been described as having beneficial influence in bone strength and architecture. However, negative systemic effects have been reported on oral administration of Sr ranelate, leading to strict restrictions in clinical application. We hypothesized that local delivery of Sr improves osteogenesis without eliciting detrimental side effects. Therefore, the in vivo response to an injectable Sr-hybrid system composed of RGD-alginate hydrogel cross-linked in situ with Sr and reinforced with Sr-doped hydroxyapatite microspheres, was investigated. The system was injected in a critical-sized bone defect model and compared to a similar Sr-free material. Micro-CT results show a trend towards higher new bone formed in Sr-hybrid group and major histological differences were observed between groups. Higher cell invasion was detected at the center of the defect of Sr-hybrid group after 15 days with earlier bone formation. Higher material degradation with increase of collagen fibers and bone formation in the center of the defect after 60 days was observed as opposed to bone formation restricted to the periphery of the defect in the control. These histological findings support the evidence of an improved response with the Sr enriched material. Importantly, no alterations were observed in the Sr levels in systemic organs or serum.

Fibrin functionalization with synthetic adhesive ligands interacting with α6ß1 integrin receptor enhance neurite outgrowth of embryonic stem cell-derived neural stem/progenitors.

To enhance fibrin hydrogel affinity towards pluripotent stem cell-derived neural stem/progenitor cells (NSPCs) and its capacity to support NSPC migration and neurite extension, we explored the tethering of synthetic peptides engaging integrin α6ß1, a cell receptor enriched in NSPCs. Six α6ß1 integrin ligands were tested for their ability to support integrin α6ß1-mediated adhesion of embryonic stem cell-derived NSPCs (ES-NSPs) and sustain ES-NSPC viability, migration, and neuronal differentiation. Due to their better performance, peptides T1, HYD1, and A5G81 were immobilized into fibrin and functionalized gels characterized in terms of peptide binding efficiency, structure and viscoelastic properties. Tethering of T1 or HYD1 successfully enhanced cell outgrowth from ES-NSPC neurospheres (up to 2.4-fold increase), which exhibited a biphasic response to peptide concentration. Inhibition assays evidenced the involvement of α6ß1 and α3ß1 integrins in mediating radial outgrowth on T1-/HYD1-functionalized gels. Fibrin functionalization also promoted neurite extension of single ES-NSPCs in fibrin, without affecting cell proliferation and neuronal differentiation. Finally, HYD1-functionalized gels were found to provide a permissive environment for axonal regeneration, leading up to a 2.0-fold increase in neurite extension from rat dorsal root ganglia explants as compared to unmodified fibrin, and to significant improved locomotor function after spinal cord injury (complete transection), along with a trend toward a higher area positive for growth associated protein 43 (marker for axonal growth cone formation). Our results suggest that conjugation of α6ß1 integrin-binding motifs is of interest to increase the biofunctionality of hydrogels used in 3D platforms for ES-NSPC culture and potentially, in matrix-assisted ES-NSPC transplantation. STATEMENT OF SIGNIFICANCE: Impact statement: The transplantation of NSPCs derived from pluripotent stem cells holds much promise for the treatment of central nervous system disorders. Moreover, the combinatorial use of biodegradable hydrogels with NSPCs was shown to contribute to the establishment of a more permissive environment for survival and integration of transplanted cells. In this study, fibrin hydrogels functionalized with a synthetic peptide engaging integrin α6ß1 (HYD1) were shown to promote neurite extension of ES-NSPCs, which is fundamental for the formation of functional neuronal relay circuits after NSPC transplantation. Notably, HYD1-functionalized fibrin per se led to enhanced axonal growth ex vivo and to an improvement in locomotor function after implantation in a rat model of spinal cord injury. Conjugation of α6ß1 integrin-binding motifs may therefore be of interest to confer bioactivity to NSPC hydrogel vehicles.