Optimization of photocrosslinkable resin components and 3D printing process parameters.

The role of 3D printing in the biomedical field is growing. In this context, photocrosslink-based 3D printing procedures for resorbable polymers stand out. Despite much work, more studies are needed on photocuring stereochemistry, new resin additives, new polymers and resin components. As part of these studies it is vital to present the logic used to optimize the amount of each resin constituent and how that effects printing process parameters. The present manuscript aims to analyze the effects of poly(propylene fumarate) (PPF) resin components and their effect on 3D printing process parameters. Diethyl fumarate (DEF), bisacylphosphine oxide (BAPO), Irgacure 784, 2-hydroxy-4-methoxybenzophenone (HMB) and, for the first time, in biomedical 3D printing, ethyl acetate (EA), were the resin components under investigation in this study. Regarding printing process parameters, Exposure Time, Voxel Depth, and Overcuring Depth were the parameters studied. Taguchi Design of Experiments was used to search for the effect of varying these resin constituent concentrations and 3D printing parameters on the curing behavior of 3D printable PPF resins. Our results indicate that resins with higher polymer cross-link density, especially those with a higher content of PPF, are able to be printed at higher voxel depth and with greater success (i.e., high yield). High voxel depth, as long as it does not sacrifice required resolution, is desirable as it speeds printing. Nevertheless, the overall process is governed by the correct setup of the voxel depth in relation to overcuring depth. In regards to resin biocompatibility, it was observed that EA is more effective than DEF, the material we had previously relied on. Our preliminary in vitro cytotoxicity tests indicate that the use of EA does not reduce scaffold biocompatibility as measured by standard cytotoxicity testing (i.e., ISO 10993-5). We demonstrate a workpath for resin constituent concentration optimization through thin film tests and photocrosslinkable process optimization. STATEMENT OF SIGNIFICANCE: We report here the results of a study of photo-crosslinkable polymer resin component optimization for the 3D printing of resorbable poly(propylene fumarate) (PPF) scaffolds. Resin additives are initially optimized for PPF thin film printing. Once those parameters have been optimized the 3D printing process parameters for PPF objects with complex, porous shapes can be optimized. The design of experiments to optimize both polymer thin films and complex porous resorbable polymer scaffolds is important as a guess and check, or in some cases a systematic method, are very likely to be too time consuming to accomplish. Previously unstudied resin components and process parameters are reported.

Poly(anhydride-ester) and poly(N-vinyl-2-pyrrolidone) blends: salicylic acid-releasing blends with hydrogel-like properties that reduce inflammation.

Polymers such as poly(N-vinyl-2-pyrrolidone) (PVP) have been used to prepare hydrogels for wound dressing applications but are not inherently bioactive. For enhanced healing, PVP was blended with salicylic acid-based poly(anhydride-esters) (SAPAE) and shown to exhibit hydrogel properties upon swelling. In vitro release studies demonstrated that the chemically incorporated drug (SA) was released from the polymer blends over 3-4 d in contrast to 3 h, and that blends of higher PVP content displayed greater swelling values and faster SA release. The polymer blends significantly the inflammatory cytokine, TNF-α, in vitro without negative effects.

Biosynthesis of N,N-dimethyltryptamine (DMT) in a melanoma cell line and its metabolization by peroxidases.

Tryptophan (TRP) is essential for many physiological processes, and its metabolism changes in some diseases such as infection and cancer. The most studied aspects of TRP metabolism are the kynurenine and serotonin pathways. A minor metabolic route, tryptamine and N,N-dimethyltryptamine (DMT) biosynthesis, has received far less attention, probably because of the very low amounts of these compounds detected only in some tissues, which has led them to be collectively considered as trace amines. In a previous study, we showed a metabolic interrelationship for TRP in melanoma cell lines. Here, we identified DMT and N,N-dimethyl-N-formyl-kynuramine (DMFK) in the supernatant of cultured SK-Mel-147 cells. Furthermore, when we added DMT to the cell culture, we found hydroxy-DMT (OH-DMT) and indole acetic acid (IAA) in the cell supernatant at 24 h. We found that SK-Mel-147 cells expressed mRNA for myeloperoxidase (MPO) and also had peroxidase activity. We further found that DMT oxidation was catalyzed by peroxidases. DMT oxidation by horseradish peroxidase, H2O2 and MPO from PMA-activated neutrophils produced DMFK, N,N-dimethyl-kynuramine (DMK) and OH-DMT. Oxidation of DMT by peroxidases apparently uses the common peroxidase cycle involving the native enzyme, compound I and compound II. In conclusion, this study describes a possible alternative metabolic pathway for DMT involving peroxidases that has not previously been described in humans and identifies DMT and metabolites in a melanoma cell line. The extension of these findings to other cell types and the biological effects of DMT and its metabolites on cell proliferation and function are key questions for future studies.

High concentrations of the melatonin metabolite, N1-acetyl-N2-formyl-5-methoxykynuramine, in cerebrospinal fluid of patients with meningitis: a possible immunomodulatory mechanism.

We evaluated the presence of the melatonin metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), in cerebrospinal fluid (CSF) of patients with viral meningitis (n = 20) and control samples (n = 8) and correlate AFMK levels with inflammatory markers such as cellularity, protein, tumor necrosis factor (TNF)-alpha, interleukin (IL)-8 and IL-1beta levels. A portion of the CSF was extracted with dichloromethane (1:5) and analyzed by high-performance liquid chromatography (HPLC) under standardized conditions for AFMK. AFMK was detected in 16 of 20 CSF samples of patients with viral meningitis; the concentration of AFMK was found to be above the quantification limit (50 nmol/L) in six of these samples. AFMK was not detected in any of the eight control samples. The samples were classified into groups according to AFMK levels: undetectable (<10 nmol/L, group I), detectable but below the quantification limit (< 50 nmol/L, group II), and quantified (>50 nmol/L, group III). Group II presented the highest levels of proteins and IL-8, whereas group III showed the lowest levels of the inflammatory parameters. This study supports our hypothesis that inflammation favors the formation of AFMK and that this compound has immunomodulatory activity in vivo.

Assessment of monocytic component in acute myelomonocytic and monocytic/monoblastic leukemias by a chemiluminescent assay.

INTRODUCTION: Classically, the monocytic component of acute myelomonocytic (FAB-M4) and acute monocytic/monoblastic (FAB-M5) leukemias is demonstrated by nonspecific esterase positivity in cytochemical stainings. We have previously demonstrated that non-specific esterases from normal monocytes can be determined by a chemiluminescent method. In the present study, we investigated whether this assay can also determine the monocytic component of FAB-M4 and FAB-M5 and distinguish these acute myeloid leukemia (AML) categories. MATERIALS AND METHODS: Bone marrow samples were obtained from 66 patients with AML (M0, two cases; M1, 12 cases; M2, 13 cases; M3, 10 cases; M4, 11 cases; M5, 12 cases; M6, two cases; M7, four cases). Cells were incubated with a standard reaction mixture and chemiluminescence was measured for 10 min. Two parameters were assessed, the peak (PLE) and the integrated light emission (ILE). RESULTS: Both PLE and ILE were higher in FAB-M4 and FAB-M5 subtypes compared to other AML subtypes (P<0.001). In addition, the classification of AML cases into FAB-M4, FAB-M5 and nonmonocytic subtypes based on ILE analysis was concordant with alpha-naphthyl acetate esterase (ANAE) in 97% of cases (kappa coefficient 0.94, P<0.001). CONCLUSIONS: These findings indicate that this chemiluminescent assay was able to determine the monocytic component of FAB-M4 and FAB-M5 cells, and the classification of AML subtypes based on chemiluminescent analysis strongly agreed with the cytochemical ANAE-staining. In conclusion, this chemiluminescent assay is a simple, fast and objective method, which may be useful as an alternative tool in the differential diagnosis of AML subtypes.

Singlet molecular oxygen: generation, reactivity, identification and biological effects

Electronically excited singlet molecular oxygen ((1)O2)) is of great importance in chemical and biological systems due to its high reactivity and involvement in physiological and pathological processes. It is a simple and useful reagent in organic synthesis of peroxides, endoperoxides, hydroperoxides, and dioxetanes. In biological systems, (1)O2 has been implicated in: i) Defense mechanisms of living organisms such as phagocytosis; ii) degradative oxidation of endogenous hallucinogens; iii) hormonal activity of prostaglandins; iv) photochemotherapy utilizing the photodynamic action of synthetic dyes; v) photosensitivity to drugs like chlorpromazine and vi) inborn errors of metabolism exemplified by erythropoietic porphyria. The high reactivity of (1)O2 with unsaturated compounds, sulfides and amines arises from its high electrophilicity and relatively long lifetime (2-4 ms in H2O and ~700 ms in CCI4). Thus, biological targets for (1)O2 having the above functional groups include unsaturated fatty acids, proteins, and DNA. Extensively conjugated biomolecules such as carotenoids act as chemical and physical quenchers of (1)O2 and hence provide protective mechanisms against the deleterious effects of this excited state of molecular oxygen. However, due to the difficulties involved in obtaining (1)O2 free from other reactive contaminants, there is a paucity of detailed studies on the mentioned aspects of (1)O2 biochemistry. Chemical and dye-sensitized photophysical methods are available to prepare (1)O2. The aim of this work is to give a general view on (1)O2 with regard to its chemical generation, reactivity with biologically important compounds, detection and its role in biological systems.

Enolizable carbonyl and imino metabolites may act as endogenous sources of reactive oxygen species

Highly reactive oxyradicals and electronically excited triplet carbonyls can be generated in vitro by iron complexes and heme enzyme-catalyzed aerobic oxidation of synthetic or naturally occurring substances capable of enolization in aqueous medium. Monoenols and enamines, obtained by (alpha-methyne-carbonyl and -imine enolization, undergo dioxygen insertion and ultimately originate triplet species; e.g., isobutanal, 3-methylacetoacetone, Schiff bases. In turn, (alpha-hydroxy- and (alpha-aminocarbonyls (e.g., carbohydrates, 5-aminolevulinic acid) tautomerize to enediols and enolamines and yield oxyradicals, initiated by electron transfer to dioxygen, as polyphenols (e.g., 6-hydroxydopamine) and polyphenolamines do. Free radicals and excited species have been implicated in several normal and pathological processes. We here briefly review our contributions to this research area, emphasizing a possible in vivo prooxidant role for 5-aminolevulinic acid, the heme precursor accumulated in several porphyric disorders (e.g., lead poisoning, acut intermittent porphyria, tyrosinosis).