Injectable Magnetic-Responsive Short-Peptide Supramolecular Hydrogels: Ex Vivo and In Vivo Evaluation.

The inclusion of magnetic nanoparticles (MNP) in a hydrogel matrix to produce magnetic hydrogels has broadened the scope of these materials in biomedical research. Embedded MNP offer the possibility to modulate the physical properties of the hydrogel remotely and on demand by applying an external magnetic field. Moreover, they enable permanent changes in the mechanical properties of the hydrogel, as well as alterations in the micro- and macroporosity of its three-dimensional (3D) structure, with the associated potential to induce anisotropy. In this work, the behavior of biocompatible and biodegradable hydrogels made with Fmoc-diphenylalanine (Fmoc-FF) (Fmoc = fluorenylmethoxycarbonyl) and Fmoc-arginine-glycine-aspartic acid (Fmoc-RGD) short peptides to which MNP were incorporated was studied in detail with physicochemical, mechanical, and biological methods. The resulting hybrid hydrogels showed enhance mechanical properties and withstood injection without phase disruption. In mice, the hydrogels showed faster and improved self-healing properties compared to their nonmagnetic counterparts. Thanks to these superior physical properties and stability during culture, they can be used as 3D scaffolds for cell growth. Additionally, magnetic short-peptide hydrogels showed good biocompatibility and the absence of toxicity, which together with their enhanced mechanical stability and excellent injectability make them ideal biomaterials for in vivo biomedical applications with minimally invasive surgery. This study presents a new approach to improving the physical and mechanical properties of supramolecular hydrogels by incorporating MNP, which confer structural reinforcement and stability, remote actuation by magnetic fields, and better injectability. Our approach is a potential catalyst for expanding the biomedical applications of supramolecular short-peptide hydrogels.

In vivo time-course biocompatibility assessment of biomagnetic nanoparticles-based biomaterials for tissue engineering applications.

Novel artificial tissues with potential usefulness in local-based therapies have been generated by tissue engineering using magnetic-responsive nanoparticles (MNPs). In this study, we performed a comprehensive in vivo characterization of bioengineered magnetic fibrin-agarose tissue-like biomaterials. First, in vitro analyses were performed and the cytocompatibility of MNPs was demonstrated. Then, bioartificial tissues were generated and subcutaneously implanted in Wistar rats and their biodistribution, biocompatibility and functionality were analysed at the morphological, histological, haematological and biochemical levels as compared to injected MNPs. Magnetic Resonance Image (MRI), histology and magnetometry confirmed the presence of MNPs restricted to the grafting area after 12 weeks. Histologically, we found a local initial inflammatory response that decreased with time. Structural, ultrastructural, haematological and biochemical analyses of vital organs showed absence of damage or failure. This study demonstrated that the novel magnetic tissue-like biomaterials with improved biomechanical properties fulfil the biosafety and biocompatibility requirements for future clinical use and support the use of these biomaterials as an alternative delivery route for magnetic nanoparticles.

Evaluation of Fibrin-Agarose Tissue-Like Hydrogels Biocompatibility for Tissue Engineering Applications.

Generation of biocompatible and biomimetic tissue-like biomaterials is crucial to ensure the success of engineered substitutes in tissue repair. Natural biomaterials able to mimic the structure and composition of native extracellular matrices typically show better results than synthetic biomaterials. The aim of this study was to perform an in vivo time-course biocompatibility analysis of fibrin-agarose tissue-like hydrogels at the histological, imagenological, hematological, and biochemical levels. Tissue-like hydrogels were produced by a controlled biofabrication process allowing the generation of biomechanically and structurally stable hydrogels. The hydrogels were implanted subcutaneously in 25 male Wistar rats and evaluated after 1, 5, 9, and 12 weeks of in vivo follow-up. At each period of time, animals were analyzed using magnetic resonance imaging (MRI), hematological analyses, and histology of the local area in which the biomaterials were implanted, along with major vital organs (liver, kidney, spleen, and regional lymph nodes). MRI results showed no local or distal alterations during the whole study period. Hematology and biochemistry showed some fluctuation in blood cells values and in some biochemical markers over the time. However, these parameters were progressively normalized in the framework of the homeostasis process. Histological, histochemical, and ultrastructural analyses showed that implantation of fibrin-agarose scaffolds was followed by a progressive process of cell invasion, synthesis of components of the extracellular matrix (mainly, collagen) and neovascularization. Implanted biomaterials were successfully biodegraded and biointegrated at 12 weeks without any associated histopathological alteration in the implanted zone or distal vital organs. In summary, our in vivo study suggests that fibrin-agarose tissue-like hydrogels could have potential clinical usefulness in engineering applications in terms of biosafety and biocompatibility.

No association between G1359A CB1 polymorphisms and pain in young northeastern Mexicans.

AIM: Recent studies show an association between the endocannabinoid system and pain. In this study, we analyzed the association between two CNR1 gene polymorphisms and pain perception in a northeast Mexican population. METHODS: Genotypic and allelic frequencies were obtained for both polymorphisms. Pain threshold, tolerance and perception were measured using the cold pressor task. RESULTS: No significant association between the polymorphisms and pain perception was found (p > 0.05). CONCLUSION: Genotypic and allelic frequencies for both polymorphisms were reported for the first time in a Mexican population; however, our results suggest that there is not a significant association between these and pain.

Generation of genipin cross-linked fibrin-agarose hydrogel tissue-like models for tissue engineering applications.

The generation of biomimetic and biocompatible artificial tissues is the basic research objective for tissue engineering (TE). In this sense, the biofabrication of scaffolds that resemble the tissues' extracellular matrix is an essential aim in this field. Uncompressed and nanostructured fibrin-agarose hydrogels (FAH and NFAH, respectively) have emerged as promising scaffolds in TE, but their structure and biomechanical properties must be improved in order to broaden their TE applications. Here, we generated and characterized novel membrane-like models with increased structural and biomechanical properties based on the chemical cross-linking of FAH and NFAH with genipin (GP at 0.1%, 0.25%, 0.5% and 0.75%). Furthermore, the scaffolds were subjected to rheological (G, G', G″ modulus), ultrastructural and ex vivo biocompatibility analyses. Results showed that all GP concentrations increased the stiffness (G) and especially the elasticity (G') of FAH and NFAH. Ultrastructural analyses demonstrated that GP and nanostructuration of FAH allowed us to control the porosity of FAH. In addition, biological studies revealed that higher concentration of GP (0.75%) started to compromise the cell function and viability. Finally, this study demonstrated the possibility to generate natural and biocompatible FAH and NFAH with improved structural and biomechanical properties by using 0.1%-0.5% of GP. However, further in vivo studies are needed in order to demonstrate the biocompatibility, biodegradability and regeneration capability of these cross-linked scaffolds.

Histological transformations of the dental pulp as possible indicator of post mortem interval: a pilot study.

BACKGROUND: The correct estimation of the post mortem interval (PMI) can be crucial on the success of a forensic investigation. Diverse methods have been used to estimate PMI, considering physical changes that occur after death, such as mortis algor, livor mortis, among others. Degradation after death of dental pulp is a complex process that has not yet been studied thoroughly. It has been described that pulp RNA degradation could be an indicator of PMI, however this study is limited to 6 days. The tooth is the hardest organ of the human body, and within is confined dental pulp. The pulp morphology is defined as a lax conjunctive tissue with great sensory innervation, abundant microcirculation and great presence of groups of cell types. AIM: The aim of this study is to describe the potential use of pulp post mortem alterations to estimate PMI, using a new methodology that will allow obtainment of pulp tissue to be used for histomorphological analysis. The current study will identify potential histological indicators in dental pulp tissue to estimate PMI in time intervals of 24h, 1 month, 3 months and 6 months. MATERIALS AND METHOD: This study used 26 teeth from individuals with known PMI of 24h, 1 month, 3 months or 6 months. All samples were manipulated with the new methodology (Carrasco, P. and Inostroza C. inventors; Universidad de los Andes, assignee. Forensic identification, post mortem interval estimation and cause of death determination by recovery of dental tissue. United State patent US 61/826,558 23.05.2013) to extract pulp tissue without the destruction of the tooth. The dental pulp tissues obtained were fixed in formalin for the subsequent generation of histological sections, stained with Hematoxylin Eosin and Masson's Trichrome. All sections were observed under an optical microscope using magnifications of 10× and 40×. RESULTS: The microscopic analysis of the samples showed a progressive transformation of the cellular components and fibers of dental pulp along PMI. These results allowed creating a chart of qualitative and quantitative parameters to be used on the estimation on PMI based on microscopic degradation of dental pulp. CONCLUSIONS: The histological transformations of dental pulp as a function of time can be used as PMI indicators.

Biocompatible magnetic core-shell nanocomposites for engineered magnetic tissues.

The inclusion of magnetic nanoparticles into biopolymer matrixes enables the preparation of magnetic field-responsive engineered tissues. Here we describe a synthetic route to prepare biocompatible core-shell nanostructures consisting of a polymeric core and a magnetic shell, which are used for this purpose. We show that using a core-shell architecture is doubly advantageous. First, gravitational settling for core-shell nanocomposites is slower because of the reduction of the composite average density connected to the light polymer core. Second, the magnetic response of core-shell nanocomposites can be tuned by changing the thickness of the magnetic layer. The incorporation of the composites into biopolymer hydrogels containing cells results in magnetic field-responsive engineered tissues whose mechanical properties can be controlled by external magnetic forces. Indeed, we obtain a significant increase of the viscoelastic moduli of the engineered tissues when exposed to an external magnetic field. Because the composites are functionalized with polyethylene glycol, the prepared bio-artificial tissue-like constructs also display excellent ex vivo cell viability and proliferation. When implanted in vivo, the engineered tissues show good biocompatibility and outstanding interaction with the host tissue. Actually, they only cause a localized transitory inflammatory reaction at the implantation site, without any effect on other organs. Altogether, our results suggest that the inclusion of magnetic core-shell nanocomposites into biomaterials would enable tissue engineering of artificial substitutes whose mechanical properties could be tuned to match those of the potential target tissue. In a wider perspective, the good biocompatibility and magnetic behavior of the composites could be beneficial for many other applications.

Generation and Characterization of Novel Magnetic Field-Responsive Biomaterials.

We report the preparation of novel magnetic field-responsive tissue substitutes based on biocompatible multi-domain magnetic particles dispersed in a fibrin-agarose biopolymer scaffold. We characterized our biomaterials with several experimental techniques. First we analyzed their microstructure and found that it was strongly affected by the presence of magnetic particles, especially when a magnetic field was applied at the start of polymer gelation. In these samples we observed parallel stripes consisting of closely packed fibers, separated by more isotropic net-like spaces. We then studied the viability of oral mucosa fibroblasts in the magnetic scaffolds and found no significant differences compared to positive control samples. Finally, we analyzed the magnetic and mechanical properties of the tissue substitutes. Differences in microstructural patterns of the tissue substitutes correlated with their macroscopic mechanical properties. We also found that the mechanical properties of our magnetic tissue substitutes could be reversibly tuned by noncontact magnetic forces. This unique advantage with respect to other biomaterials could be used to match the mechanical properties of the tissue substitutes to those of potential target tissues in tissue engineering applications.

Cryopreservation of an artificial human oral mucosa stroma. A viability and rheological study.

The aim of this study was to evaluate the viability and biomechanical properties of artificial human oral mucosa stroma (HOMS) subjected to cryopreservation with different cryoprotectant solutions. Artificial HOMS based on a fibrin-agarose matrix with human gingival fibroblasts cultured 7 days in vitro were cryopreserved with three cryoprotectant solutions: (A) TC-199 Medium, DMSO 15%, albumin; (B) DMEM, FCS, DMSO 10%; (C) QC Medium, glycerol. As controls, artificial HOMS not subjected to cryopreservation (CF) and HOMS cryopreserved without cryoprotectant solution (CS) were used. Histological analysis by light microscopy showed that solutions A and B preserved a pattern of porosity similar to values in CF. Based on the number of intact cells in the fibrin-agarose matrix, substitutes preserved with solution B showed the best results. Cell proliferation detected with PCNA immunochemical methods showed that the cell proliferation index was highest in substitutes cryopreserved with solution B. The reculture method and cell viability analyses with Live & Dead(®) revealed increased number of viable in cells preserved with solution B. Artificial stroma substitutes in CS control samples showed the greatest alterations in microstructure and cell proliferation. Analysis of the biomechanical properties showed that substitutes cryopreserved with different solutions had adequate rheological parameters (yield stress, elastic modulus and viscous modulus) and were therefore suitable for use in regenerative medicine. These results establish effective methods of cryopreservation for all experimental situations and suggest that solution B (DMEM, FCS, DMSO 10%) was the best cryoprotectant for the cryopreservation of an artificial oral human mucosa substitute based on a fibrin-agarose matrix.

An in vitro biocompatibility study of conventional and resin-modified glass ionomer cements.

PURPOSE: To evaluate the biocompatibility of a glass-ionomer (GIC) and a resin-modified glass-ionomer cement (RM-GIC), cell viability was examined in a model of human gingival fibroblasts using morphological, biochemical, and ionic patterns by means of phase contrast microscopy, lactate dehydrogenase (LDH) release, and quantitative x-ray microanalysis (EPXMA). MATERIALS AND METHODS: The GIC Ketac-Molar Easymix (3M ESPE) and the RM-GIC Vitrebond (3M ESPE) were compared in human gingival fibroblasts exposed to the cements for 72 h. As controls, fibroblasts cultured with DMEM culture medium (negative control) and with 1% triton × (positive control) were used. RESULTS: Light microscopic findings showed greater morphological alterations in cells exposed to RM-GIC than to GIC. The relative percentage of LDH released from the cells to the supernatant was significantly higher in RMGIC cultures than in the control. Quantitative x-ray microanalysis showed that cultures exposed to RM-GIC were characterized by an increase in intracellular Na and a decrease in intracellular Cl and K. These changes in ion composition were significant compared to control and GIC cultures. CONCLUSION: The three indicators of cellular biocompatibility after 72 h of exposure showed that RM-GIC led to more marked alterations than GIC in human gingival fibroblasts.

Biological evaluation of 2-hydroxyethylmethacrylate (HEMA) toxicity in human gingival fibroblasts with histochemical X-ray microanalysis.

PURPOSE: To analyze the cytotoxic effects of 2-hydroxyethylmethacrylate (HEMA) in human gingival fibroblasts using quantitative x-ray microanalysis (EXPMA) and two classical methods (DNA and LDH release in culture medium). MATERIALS AND METHODS: Different concentrations of HEMA (5, 10, 20, 30, and 40 mM) in DMEM medium were used and the effects on human gingival fibroblasts after 6, 12, and 24 h were determined. As controls, fibroblasts cultured with DMEM culture medium (negative control) and fibroblast incubated in 1% triton X (positive control) were used. RESULTS: The results showed that correlation between the concentrations of HEMA and the amount of LDH and DNA released to the medium were statistically significant for all times analyzed. LDH and DNA released from cells incubated in the lowest concentrations of HEMA (5 and 10 mM) were not significantly different to negative controls. In contrast, cells incubated in the highest HEMA concentrations (20, 30, 40 mM) showed a significant increase of both LDH and DNA released to the culture medium at 6, 12, and 24 h. On the other hand, the ionic concentration of the different elements analyzed in this work revealed that the contents of P, S, Cl, and K were significantly higher in the controls than in samples incubated for 6 h in 5 mM or 10 mM HEMA (p < 0.01). K/ Na index (an excellent marker of cell viability) showed a significant decrease, and therefore, viability was significantly reduced. CONCLUSION: The results suggest that EXPMA is a sensitive method that is able to detect early cell damage even before the cell membrane is altered.

Hybrid cell death induced by exposure to 2-hydroxyethyl methacrylate (HEMA): an ultrastructural and X-ray microanalytical study.

PURPOSE: The aim of this study was to evaluate the ultrastructural characteristics and ionic profile of U937 cells after exposure to 2-hydroxyethyl methacrylate (HEMA) to shed light on the cytotoxicity of this dental adhesive and its relation to mechanisms of cell death. MATERIALS AND METHODS: U937 human monoblastic cells were incubated in RPMI 1640 culture medium and exposed to HEMA at LD50. Structural changes after 5, 15, 30, 60, and 120 min were observed with transmission electron microscopy. Ionic content of Na, K, Cl, Mg, P and S was evaluated by quantitative electron probe X-ray microanalysis. RESULTS: Our results in human monoblastic cell line U937 establish that exposure to HEMA at LD50 led to a singular mechanism of cell death characterized by changes in the morphology and ultrastructure of the cells (cell size, blebs, and organelle structure) compatible with apoptosis, but without changes in nuclear ultrastructure. These findings were consistent with our microanalytical data, which revealed a significant increase in intracellular Na and a decrease in K, along with a significant initial decrease in Cl concentration followed later (120 min) by an increase. CONCLUSION: All three lines of evidence (cell morphology, ultrastructural changes, and ionic profile) showed that HEMA at LD50 led to a hybrid process of cell death. We suggest that apoptosis and necrosis are part of a continuum comprising a single process of cell death.