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The regeneration of bone remains one of the main challenges in the biomedical field, with the need to provide more personalized and multifunctional solutions. The other persistent challenge is related to the local prevention of infections after implantation surgery. To fulfill the first one and provide customized scaffolds with complex geometries, 3D printing is being investigated, with polylactic acid (PLA) as the biomaterial mostly used, given its thermoplastic properties. The 3D printing of PLA in combination with hydroxyapatite (HA) is also under research, to mimic the native mechanical and biological properties, providing more functional scaffolds. Finally, to fulfill the second one, antibacterial drugs locally incorporated into biodegradable scaffolds are also under investigation. This work aims to develop vancomycin-loaded 3D-printed PLA-HA scaffolds offering a dual functionality: local prevention of infections and personalized biodegradable scaffolds with osseointegrative properties. For this, the antibacterial drug vancomycin was incorporated into 3D-printed PLA-HA scaffolds using three loading methodologies: (1) dip coating, (2) drop coating, and (3) direct incorporation in the 3D printing with PLA and HA. A systematic characterization was performed, including release kinetics, Staphylococcus aureus antibacterial/antibiofilm activities and cytocompatibility. The results demonstrated the feasibility of the vancomycin-loaded 3D-printed PLA-HA scaffolds as drug-releasing vehicles with significant antibacterial effects for the three methodologies. In relation to the drug release kinetics, the (1) dip- and (2) drop-coating methodologies achieved burst release (first 60 min) of around 80-90% of the loaded vancomycin, followed by a slower release of the remaining drug for up to 48 h, while the (3) 3D printing presented an extended release beyond 7 days as the polymer degraded. The cytocompatibility of the vancomycin-loaded scaffolds was also confirmed.

Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes in the plant genome. Little is known about how this small set is linked to their hormone profile, which determines development, morphology, and levels of secondary metabolite production. We used C. asiatica hairy root line cultures to determine the putative links between the rol and aux gene expressions with morphological traits, a hormone profile, and centelloside production. The results obtained after 14 and 28 days of culture were processed via multivariate analysis and machine-learning processes such as random forest, supported vector machines, linear discriminant analysis, and neural networks. This allowed us to obtain models capable of discriminating highly productive root lines from their levels of genetic expression ( rol and aux genes) or from their hormone profile. In total, 12 hormones were evaluated, resulting in 10 being satisfactorily detected. Within this set of hormones, abscisic acid (ABA) and cytokinin isopentenyl adenosine (IPA) were found to be critical in defining the morphological traits and centelloside content. The results showed that IPA brings more benefits to the biotechnological platform. Additionally, we determined the degree of influence of each of the evaluated genes on the individual hormone profile, finding that aux1 has a significant influence on the IPA profile, while the rol genes are closely linked to the ABA profile. Finally, we effectively verified the gene influence on these two specific hormones through feeding experiments that aimed to reverse the effect on root morphology and centelloside content., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Alcalde, Müller, Munné-Bosch, Landín, Gallego, Bonfill, Palazon and Hidalgo-Martinez.)

Main Conclusion: Shoot tip necrosis is a physiological condition that negatively impacts the growth and development of in vitro plant shoot cultures across a wide range of species. Shoot tip necrosis is a physiological condition and disorder that can arise in plantlets or shoots in vitro that results in death of the shoot tip. This condition, which can spread basipetally and affect the emergence of axillary shoots from buds lower down the stem, is due to the cessation of apical dominance. STN can occur at both shoot multiplication and rooting stages. One of the most common factors that cause STN is nutrient deficiency or imbalance. Moreover, the presence or absence of plant growth regulators (auxins or cytokinins) at specific developmental stages may impact STN. The cytokinin to auxin ratio within an in vitro plant can be modified by varying the concentration of cytokinins used in the culture medium. The supply of nutrients to in vitro shoots or plantlets might also affect their hormonal balance, thus modifying the occurrence of STN. High relative humidity within culture vessels and hyperhydricity are associated with STN. An adequate supply of calcium as the divalent cation (Ca 2+ ) can hinder STN by inhibiting the accumulation of phenolic compounds and thus programmed cell death. Moreover, the level of Ca 2+ affects auxin transport and ethylene production, and higher ethylene production, which can occur as a result of high relative humidity in or poor ventilation of the in vitro culture vessel, induces STN. High relative humidity can decrease the mobility of Ca 2+ within a plant, resulting in Ca 2+ deficiency and STN. STN of in vitro shoots or plantlets can be halted or reversed by altering the basal medium, mainly the concentration of Ca 2+ , adjusting the levels of auxins or cytokinins, or modifying culture conditions. This review examines the literature related to STN, seeks to discover the associated factors and relations between them, proposes practical solutions, and attempts to better understand the mechanism(s) underlying this condition in vitro.

Polysaccharide-based hydrogel particles (PbHPs) are very promising carriers aiming to control and target the release of drugs with different physico-chemical properties. Such delivery systems can offer benefits through the proper encapsulation of many drugs (non-steroidal and steroidal anti-inflammatory drugs, antibiotics, etc) ensuring their proper release and targeting. This review discusses the different phases involved in the production of PbHPs in pharmaceutical technology, such as droplet formation (SOL phase), sol-gel transition of the droplets (GEL phase) and drying, as well as the different methods available for droplet production with a special focus on prilling technique. In addition, an overview of the various droplet gelation methods with particular emphasis on ionic cross-linking of several polysaccharides enabling the formation of particles with inner highly porous network or nanofibrillar structure is given. Moreover, a detailed survey of the different inner texture, in xerogels, cryogels or aerogels, each with specific arrangement and properties, which can be obtained with different drying methods, is presented. Various case studies are reported to highlight the most appropriate application of such systems in pharmaceutical field. We also describe the challenges to be faced for the breakthrough towards clinic studies and, finally, the market, focusing on the useful approach of safety-by-design (SbD)., Competing Interests: The authors declare no conflict of interest.

Organogenesis constitutes the biological feature driving plant in vitro regeneration, in which the role of plant hormones is crucial. The use of machine learning (ML) technology stands out as a novel approach to characterize the combined role of two phytohormones, the auxin indoleacetic acid (IAA) and the cytokinin 6-benzylaminopurine (BAP), on the in vitro organogenesis of unexploited medicinal plants from the Bryophyllum subgenus. The predictive model generated by neurofuzzy logic, a combination of artificial neural networks (ANNs) and fuzzy logic algorithms, was able to reveal the critical factors affecting such multifactorial process over the experimental dataset collected. The rules obtained along with the model allowed to decipher that BAP had a pleiotropic effect on the Bryophyllum spp., as it caused different organogenetic responses depending on its concentration and the genotype, including direct and indirect shoot organogenesis and callus formation. On the contrary, IAA showed an inhibiting role, restricted to indirect shoot regeneration. In this work, neurofuzzy logic emerged as a cutting-edge method to characterize the mechanism of action of two phytohormones, leading to the optimization of plant tissue culture protocols with high large-scale biotechnological applicability., Competing Interests: The authors declare no conflict of interest.

We combined machine learning and plant in vitro culture methodologies as a novel approach for unraveling the phytochemical potential of unexploited medicinal plants . In order to induce phenolic compound biosynthesis, the in vitro culture of three different species of Bryophyllum under nutritional stress was established. To optimize phenolic extraction, four solvents with different MeOH proportions were used, and total phenolic content (TPC), flavonoid content (FC) and radical-scavenging activity (RSA) were determined. All results were subjected to data modeling with the application of artificial neural networks to provide insight into the significant factors that influence such multifactorial processes. Our findings suggest that aerial parts accumulate a higher proportion of phenolic compounds and flavonoids in comparison to roots. TPC was increased under ammonium concentrations below 15 mM, and their extraction was maximum when using solvents with intermediate methanol proportions (55-85%). The same behavior was reported for RSA, and, conversely, FC was independent of culture media composition, and their extraction was enhanced using solvents with high methanol proportions (>85%). These findings confer a wide perspective about the relationship between abiotic stress and secondary metabolism and could serve as the starting point for the optimization of bioactive compound production at a biotechnological scale., Competing Interests: The authors declare no conflict of interest.

Bioceramic scaffolds are crucial in tissue engineering for bone regeneration. They usually provide hierarchical porosity, bioactivity, and mechanical support supplying osteoconductive properties and allowing for 3D cell culture. In the case of age-related diseases such as osteoarthritis and osteoporosis, or other bone alterations as alveolar bone resorption or spinal fractures, functional tissue recovery usually requires the use of grafts. These bone grafts or bone void fillers are usually based on porous calcium phosphate grains which, once disposed into the bone defect, act as scaffolds by incorporating, to their own porosity, the intergranular one. Despite their routine use in traumatology and dental applications, specific graft requirements such as osteoinductivity or balanced dissolution rate are still not completely fulfilled. Marine origin bioceramics research opens the possibility to find new sources of bone grafts given the wide diversity of marine materials still largely unexplored. The interest in this field has also been urged by the limitations of synthetic or mammalian-derived grafts already in use and broadly investigated. The present review covers the current stage of major marine origin bioceramic grafts for bone tissue regeneration and their promising properties. Both products already available on the market and those in preclinical phases are included. To understand their clear contribution to the field, the main clinical requirements and the current available biological-derived ceramic grafts with their advantages and limitations have been collected.

During the in vitro culture of plants some physiological disorders caused major problems that have been associated with culture media composition. The objective of this study was to better understand the abnormal physiological response of two pistachio rootstocks to changes in culture media ingredients. On this purpose, two computer-based tools were employed: design of experiment (DOE) and neurofuzzy logic. DOE was employed to generate a five-dimensional IV-design spaces allowing to reduce the number of treatments from 6,250 to 61. The second one, an artificial intelligence (AI) tool, neurofuzzy logic, was used to understand the cause-effect relationships between the factors studied (25) and seven physiological disorders including shoot-tip necrosis (STN), leaf necrosis (LN), leaf color (LC), basal callus (BC) formation, shoot fasciation (SF), hyperhydricity and epinasty, typically described during pistachio in vitro culture. Four out of the seven disorders were successfully modeled, being significantly affected by a limited number of factors. STN and BC were significantly affected by the concentration of EDTA - . However, while a low concentration of EDTA - reduces the STN, promotes BC. LN and LC were strongly alleviated by high amounts of thiamine-HCl. Undoubtedly, the results demonstrate the importance of recording and using data related to physiological disorders along with growth parameters when developing suitable culture media for plant tissues. The computer-based tools have been useful to: i) well sample experimental design; ii) reduce the final number of treatments and the experimental work; iii) identify the key factors affecting each disorder; iv) get insight about the causes that promote the appearance of physiological disorders. Our findings demonstrate that the recently AI designed POM media, although not optimal, is the most suitable (favouring growth and limiting physiological abnormalities) media for in vitro culture of pistachio compared to those media, currently used.

The aim of this study was to determine the effects of Murashige and Skoog (MS) salts on optimal growth of two pistachio rootstocks, P. vera cv. "Ghazvini" and "UCB1" using design of experiments (DOE) and artificial intelligence (AI) tools. MS medium with 14 macro-and micro-elements was used as base point and its concentration varied from 0 to 5 × MS concentrations. Design of experiments (DOE) software was used to generate a five-dimensional design space by categorizing MS salts into five independent factors (NH 4 NO 3 , KNO 3 , mesos, micros and iron), reducing the experimental design space from 3,125 to just 29 treatments. Typical plant growth parameters such as shoot quality (SQ), proliferation rate (PR), shoot length (SL), and some physiological disorders including shoot-tip necrosis (STN) and callus formation at the base of explants (BC) were evaluated for each treatment. The results were successfully modeled using neurofuzzy logic software. The model delivered new insights, by different sets of "IF-THEN" rules, pinpointing the key role of some ion interactions ( SO 4 2 - × Cl - , K + × SO 4 2 - × EDTA - , and Fe 2+ × Cu 2+ × NO 3 - ) for SQ, PR, and SL, whilst physiological disorders (STN and BC) were governed mainly by independent ions as Fe 2+ and EDTA - , respectively. In our opinion, the methodology and results obtained in this study is extremely useful to understand the effect of mineral nutrients on pistachio in vitro culture, through discovering new complex interactions among macro-and micro-elements which can be implemented to design new media of plant tissue culture and improve healthy plant micropropagation for any plant species.

Numerous studies have been focused on hydrophobic compounds encapsulation as oils. In fact, oils can provide numerous health benefits as synergic ingredient combined with other hydrophobic active ingredients. However, stable microparticles for pharmaceutical purposes are difficult to achieve when commonly techniques are used. In this work, sunflower oil was encapsulated in calcium-alginate capsules by prilling technique in co-axial configuration. Core-shell beads were produced by inverse gelation directly at the nozzle using a w/o emulsion containing aqueous calcium chloride solution in sunflower oil pumped through the inner nozzle while an aqueous alginate solution, coming out from the annular nozzle, produced the beads shell. To optimize process parameters artificial intelligence tools were proposed to optimize the numerous prilling process variables. Homogeneous and spherical microcapsules with narrow size distribution and a thin alginate shell were obtained when the parameters as w/o constituents, polymer concentrations, flow rates and frequency of vibration were optimized by two commercial software, FormRules® and INForm®, which implement neurofuzzy logic and Artificial Neural Networks together with genetic algorithms, respectively. This technique constitutes an innovative approach for hydrophobic compounds microencapsulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The morphology and dimensions of bioactive materials are essential attributes to promote tissue culture. Bioactive materials with nanofibrous structure have excellent potential to be used as bone-defect fillers, since they mimic the collagen in the extracellular matrix. On the other hand, bioactive glasses with applications in regenerative medicine may present antibacterial properties, which depend on glass composition, concentration and the microorganisms tested. Likewise, their morphology may influence their antibacterial activity too. In the present work, the laser-spinning technique was used to produce bioactive glass nanofibers of two different compositions: 45S5 Bioglass® and ICIE16M, bioactive glass doped with zinc and strontium. Their antibacterial activity against Staphylococcus aureus was tested by culturing them in dynamic conditions. Bacterial growth index profiles during the first days of experiment can be explained by the variations in the pH values of the media. The bactericidal effect of the doped nanofibers at longer times is justified by the release of zinc and strontium ions. Cytotoxicity was analyzed by means of cell viability tests performed with BALB/3T3 cell line.

Background: Plant acclimation is a highly complex process, which cannot be fully understood by analysis at any one specific level (i.e. subcellular, cellular or whole plant scale). Various soft-computing techniques, such as neural networks or fuzzy logic, were designed to analyze complex multivariate data sets and might be used to model large such multiscale data sets in plant biology., Methodology and Principal Findings: In this study we assessed the effectiveness of applying neuro-fuzzy logic to modeling the effects of light intensities and sucrose content/concentration in the in vitro culture of kiwifruit on plant acclimation, by modeling multivariate data from 14 parameters at different biological scales of organization. The model provides insights through application of 14 sets of straightforward rules and indicates that plants with lower stomatal aperture areas and higher photoinhibition and photoprotective status score best for acclimation. The model suggests the best condition for obtaining higher quality acclimatized plantlets is the combination of 2.3% sucrose and photonflux of 122-130 µmol m(-2) s(-1)., Conclusions: Our results demonstrate that artificial intelligence models are not only successful in identifying complex non-linear interactions among variables, by integrating large-scale data sets from different levels of biological organization in a holistic plant systems-biology approach, but can also be used successfully for inferring new results without further experimental work.

Plant tissue growth can be regulated and controlled via culture media composition. A number of different laborious and time-consuming approaches have been used to attempt development of optimized media for a wide range of species and applications. However, plant tissue culture is a very complex task, and the identification of the influences of process factors such as mineral nutrients or plant growth regulators on a wide spectrum of growth responses cannot always well comprehended. This study employs a new approach, data mining, to uncover and integrate knowledge hidden in multiple data from plant tissue culture media formulations using apricot micropropagation databases as an example. Neurofuzzy logic technology made it possible to identify relationships among several factors (cultivars, mineral nutrients and plant growth regulators) and growth parameters (shoots number, shoots length and productivity), extracting biologically useful information from each database and combining them to create a model. The IF-THEN rule sets generated by neurofuzzy logic were completely in agreement with previous findings based on statistical analysis, but advantageously generated understandable and reusable knowledge that can be applied in future plant tissue culture media optimization., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Objectives: The purpose of this work was to study the chemical stability of the new antitumoral β-lapachone (βLAP) to determine the degradation pathway/s of the molecule and the degradation kinetics in addition to identifying several degradation products., Method: Samples of βLAP in solution were stored under conditions of darkness and illumination at 40°C at which the pseudo-first order rate constants for the βLAP degradation were determined. Furthermore, drug degraded solutions were concentrated and purified using Sephadex LH-20 and preparative thin-layer chromatography and degradation products were identified by nuclear magnetic resonance spectroscopy., Key Findings: The results revealed that βLAP shows two different degradation routes: hydrolysis in the dark and photolysis under the light. The βLAP exposure to light accelerated the drug degradation about 140 fold, compared with the samples stored in the absence of light. The hydrolysis produced hydroxylapachol as the main degradation product. The photolysis yielded phthalic acid, 6-hydroxy-3methylene-3H-isobenzofuran-1-one and a benzomacrolactone together with a complex mixture of other phthalate-derivatives such as 2-(2-carboxy-acetyl)-benzoic acid., Conclusions: This study provides useful information for the development of βLAP dosage forms, their storage, manipulation and quality control., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

This study employs artificial neural networks (ANNs) to create a model to identify relationships between variables affecting the in vitro rhizogenesis and acclimatization of two cultivars of Vitis vinifera L. Albariño and Mencía. The effects of three factors (inputs), the type of cultivar, concentration and exposure time to indolebutyric acid (IBA), on the success of in vitro rhizogenesis and acclimatization were evaluated. The developed model, using ANNs software, was assessed using a separate set of validation data and was in good agreement with the observed results. Exposure time to IBA was found to have the dominant role in influencing the height of acclimatized plantlets. ANNs can be a useful tool for modeling different complex processes and data sets, in plant tissue cultures or, more generally, in plant biology., (Copyright (c) 2010 Elsevier GmbH. All rights reserved.)

Commonly, simple mathematical models can not be used to describe exactly the biological processes due to their higher complexity. In fact, most biological interactions cannot be elucidated by a simple stepwise algorithm or a precise formula, particularly when the data are complex or noisy. ANNs allows an accurate description of those kind of biological processes in plant science, offering new advantages over traditional treatments as the possibility of a model, prediction and optimize results. Different kind of data can be analyzed using a unique and "easy to use" technology. Researchers with a high specialized mathematical background are not required and ANNs offer the possibility of achieving the whole view of the experimental study with a limited number of experiments and costs. Additionally, it is possible to add new inputs and outputs to the database to reach a new understanding.

The Faculty of Pharmacy of Santiago de Compostela was founded in 1857, but research activities did not start until the beginning of the 20th century. The new Spanish regulations promoting the experimentation at universities and the stages of professors and recently graduated students outside Spain contributed to generate a remarkable research group in a provincial university. The excessive university centralism at that time in Spain and the characteristics of the headquarters of the faculty -- a 16th century Renaissance palace -- hindered, in general, the research development. However, the scientific spirit of professors and students, and their work were imposed and they achieved an estimable status in pharmacy research in Galicia. The facilities were the staff's highest priority and it raised more than a protest. A remarkable investment in apparatuses and laboratory materials was carried out and researchers started to publish original research papers in local journals, such as the "Revista de farmacia," published by their own University. In the previous years to the Spanish Civil War (1936) the research status in drug development and the background of the staff and students were important, allowing them to join the military laboratory that Franco's army created at the faculty of pharmacy. The research work continued in a small scale and was specially focused on the production of "copy drugs" coping European specific drugs which were scarce at that time. The first third of the 20th century was the germ of the great research prestige that the Faculty of Pharmacy of Santiago de Compostela enjoys at present.

This study has investigated the utility and potential advantages of an artificial intelligence technology - neurofuzzy logic - as a modeling tool to study direct compression formulations. The modeling performance was compare with traditional statistical analysis. From results it can be stated that the normalized error obtained from neurofuzzy logic was lower. Compared to the multiple regression analysis neurofuzzy logic showed higher accuracy in prediction for the five outputs studied. Rule sets generated by neurofuzzy logic are completely in agreement with the findings based on statistical analysis and advantageously generate understandable and reusable knowledge. Neurofuzzy logic is easy and rapid to apply and outcomes provided knowledge not revealed via statistical analysis.

beta-Lapachone [betaLAP] is a novel antitumor drug, which was recently on clinical trials with promising preliminary results. Problems derived from its low water solubility, its instability in solution and its high therapeutic dose constitute some challenges for pharmaceutical researchers. The purpose of the present work is to enhance the limited dissolution rate of betaLAP by the design of particles using a solvent change precipitation process. The procedure induces the spontaneous crystalline growth of the betaLAP in the presence of a stabilizing polymer (Hydroxypropylmethylcellulose) that limits the size of the particles generated. Physicochemical characterization of microparticles and the betaLAP dissolution rate was carried out. The utility of the betaLAP microcrystals in the development of tablets with adequate dissolution properties was also stated. The procedure was optimized in order to obtain stable and homogeneous particles with a small mean particle size (approximately 3 microm) and a narrow particle size distribution. There were no differences between the drying methods evaluated (in an oven and freeze-drying) with regard to particle morphology or dissolution behaviour, which is almost instantaneous. Tablets having suitable mechanical properties were produced by dry granulation prior to compression. The compression process did not compromise betaLAP dissolution characteristics.

The objective of the present study was to evaluate the compatibility of the beta-lapachone (betaLAP), an antitumoral drug in clinical phase, with pharmaceutical excipients of common use including diluents, binders, disintegrants, lubricants and solubilising agents. Differential scanning calorimetry (DSC) was used for a first screening to find small variations in peak temperatures and/or their associated enthalpy for six drug/excipient combinations (magnesium stearate, sodium estearyl fumarate, dicalcium phosphate dihydrate, mannitol, randomized methyl-beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin), which indicate some degree of interaction. Additional studies using Fourier transformed infrared spectroscopy (FTIR), optical microscopy (OM) and heating-cooling DSC (HC-DSC) confirmed the incompatibility of betaLAP with magnesium stearate and dicalcium phosphate dihydrate. Those excipients should be avoided in the development of solid dosage forms.

The purpose of this research was to explore the utility of beta cyclodextrin (betaCD) and beta cyclodextrin derivatives (hydroxypropyl-beta-cyclodextrin [HPbetaCD], sulfobutylether-beta-CD [SBbetaCD], and a randomly methylated-beta-CD [RMbetaCD]) to form inclusion complexes with the antitumoral drug, beta-lapachone (betaLAP), in order to overcome the problem of its poor water solubility. RMbetaCD presented the highest efficiency for betaLAP solubilization and was selected to develop solid-state binary systems. Differential scanning calorimetry (DSC), X-ray powder diffractometry (XRPD), Fourier transform infrared (FTIR) and optical and scanning electron microscopy results suggest the formation of inclusion complexes by both freeze-drying and kneading techniques with a dramatic improvement in drug dissolution efficiency at 20-minute dissolution efficiency (DE(20-minute) 67.15% and 88.22%, respectively) against the drug (DE(20-minute) 27.11%) or the betaCD/drug physical mixture (DE(20-minute) 27.22%). However, the kneading method gives a highly crystalline material that together with the adequate drug dissolution profile make it the best procedure in obtaining inclusion complexes of RMbetaCD/betaLAP convenient for different applications of betaLAP.

Three variables of the tablet manufacturing process (duration of mixing with lubricant, maximum compression force and compression rate) were studied for their effects on various properties of direct compression phenobarbital-microcrystalline cellulose tablets. Mixing time and maximum compression force were found to have a marked influence on friability and dissolution rate; crushing strength was found to be a useful parameter for quality control.