Bone tissue provides support and protection to different organs and tissues. Aging and different diseases can cause a decrease in the rate of bone regeneration or incomplete healing; thus, tissue-engineered substitutes can be an acceptable alternative to traditional therapies. In the present work, we have developed an in vitro osteogenic differentiation model based on mesenchymal stem cells (MSCs), to first analyse the influence of the culture media and the origin of the cells on the efficiency of this process and secondly to extrapolate it to a 3D environment to evaluate its possible application in bone regeneration therapies. Two osteogenic culture media were used (one commercial from Stemcell Technologies and a second supplemented with dexamethasone, ascorbic acid, glycerol-2-phosphate, and BMP-2), with human cells of a mesenchymal phenotype from two different origins: adipose tissue (hADSCs) and dental pulp (hDPSCs). The expression of osteogenic markers in 2D cultures was evaluated in several culture periods by means of the immunofluorescence technique and real-time gene expression analysis, taking as reference MG-63 cells of osteogenic origin. The same strategy was extrapolated to a 3D environment of polylactic acid (PLA), with a 3 alginate hydrogel. The expression of osteogenic markers was detected in both hADSCs and hDPSCs, cultured in either 2D or 3D environments. However, the osteogenic differentiation of MSCs was obtained based on the culture medium and the cell origin used, since higher osteogenic marker levels were found when hADSCs were cultured with medium supplemented with BMP-2. Furthermore, the 3D culture used was suitable for cell survival and osteogenic induction.

Several tissue engineering stem cell-based procedures improve hyaline cartilage repair. In this work, the chondrogenic potential of dental pulp stem cell (DPSC) organoids or microtissues was studied. After several weeks of culture in proliferation or chondrogenic differentiation media, synthesis of aggrecan and type II and I collagen was immunodetected, and SOX9, ACAN, COL2A1, and COL1A1 gene expression was analysed by real-time RT-PCR. Whereas microtissues cultured in proliferation medium showed the synthesis of aggrecan and type II and I collagen at the 6^th week of culture, samples cultured in chondrogenic differentiation medium showed an earlier and important increase in the synthesis of these macromolecules after 4 weeks. Gene expression analysis showed a significant increase of COL2A1 after 3 days of culture in chondrogenic differentiation medium, while COL1A1 was highly expressed after 14 days. Cell-cell proximity promotes the chondrogenic differentiation of DPSCs and important synthesis of hyaline chondral macromolecules.

The aim of this work is to study the effect of platelet factors on the differentiation of mesenchymal stem cells (MSCs) to hyaline cartilage chondrocytes in a three-dimensional environment. MSCs were cultured in a microgel environment with a chondrogenic medium. The microgel consisted of microspheres that combine gelatin and platelet-rich plasma (PRP). The gelatin/PRP microdroplets were produced by emulsion. The gelatin containing the microdroplets was enzymatically gelled, retaining PRP and, just before seeding the cells, platelets were activated by adding calcium chloride so that platelet growth factors were released into the culture media but not before. Platelet activation was analyzed before activation to rule out the possibility that the gelatin cross-linking process itself activated the platelets. The gene expression of characteristic chondrogenic markers and miRNA expression were analyzed in cells cultured in a differentiation medium and significant differences were found between gelation/PRP microgels and those containing only pure gelatin. In summary, the gelatin microspheres effectively encapsulated platelets that secreted and released factors that significantly contributed to cellular chondrogenic differentiation. At the same time, the microgel constituted a 3D medium that provided the cells with adherent surfaces and the possibility of three-dimensional cell–cell contact.

Cell culture of bone and tendon tissues requires mechanical stimulation of the cells in order to mimic their physiological state. In the present work, a device has been conceived and developed to generate a controlled magnetic field with a homogeneous gradient in the working space. The design requirement was to maximize the magnetic flux gradient, assuring a minimum magnetizing value in a 15 mm × 15 mm working area, which highly increases the normal operating range of this sort of devices. The objective is to use the machine for two types of biological tests: magnetic irradiation of biological samples and force generation on paramagnetic particles embedded in scaffolds for cell culture. The device has been manufactured and experimentally validated by evaluating the force exerted on magnetic particles in a viscous fluid. Apart from the magnetic validation, the device has been tested for irradiating biological samples. In this case, viability of human dental pulp stem cells has been studied in vitro after electromagnetic field exposition using the designed device. After three days of irradiation treatment, cellular microtissues showed a 59% increase in the viable cell number. Irradiated cells did not show morphological differences when compared with control cells.

Climate-driven changes are reshaping the ecology of Vibrio vulnificus in European waters. Here, we present a retrospective genomic and phenotypic analysis of pre-2018 isolates belonging to lineage 4 (L4), a phylogenetic group historically confined to the Mediterranean Sea and now detected in northern Europe. Using a lineage-specific multiplex PCR combined with whole-genome sequencing, we identified 49 clinical and environmental L4 isolates from German coastal waters. Comparative genomics revealed extensive genetic plasticity in L4, indicative of frequent recombination and horizontal gene transfer, including three MARTX toxin architectures, fourteen distinct capsular genotypes, two type VI secretion systems, and multiple prophages. Notably, nearly half of the L4 isolates encoded a previously undescribed MARTX variant (type H), apparently derived from recombination within a type C toxin and containing a novel calmodulin-dependent NADase (CdN) domain with potential functional implications for virulence. One strain also harboured the plasmid-borne genes ftbp and fpcrp, which confer resistance to fish innate immunity and the ability to cause sepsis, thereby extending the distribution of the piscis pathovar to all five V. vulnificus lineages. Functional assays showed that most L4 strains withstood the bactericidal activity of iron-overloaded human serum, consistent with a capacity to cause sepsis in susceptible individuals. Collectively, these findings redefine V. vulnificus as a multi-host climate-responsive marine pathogen and establish L4 as a newly adapted European lineage whose northward expansion exemplifies how genomic diversification and ocean warming jointly drive the evolution of high-risk marine pathogens within a One Health framework.