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Tissue decellularisation has gained much attention in regenerative medicine as an alternative to synthetic materials. In decellularised tissues, biological cues can be maintained and provide cellular environments still unmet by synthetic materials. Supercritical CO2 (scCO2) has recently emerged as a promising alternative decellularisation technique to aggressive detergents; in addition, scCO2 provides innate sterilisation. However, to date, decellularisation with scCO2 is limited to only a few tissue types with low cellular density. In the current study, a scCO2 technique to decellularise high density tissues, including articular cartilage, tendon and skin, was developed. Results showed that most of the cellular material was removed, while the sample structure and biocompatibility was preserved. The DNA content was reduced in cartilage, tendon and skin as compared to the native tissue. The treatment did not affect the initial tendon elastic modulus [reduced from 126.35 ± 9.79 MPa to 113.48 ± 8.48 MPa (p > 0.05)], while it reduced the cartilage one [from 12.06 ± 2.14 MPa to 1.17 ± 0.34 MPa (p < 0.0001)]. Interestingly, cell adhesion molecules such as fibronectin and laminin were still present in the tissues after decellularisation. Bovine chondrocytes were metabolically active and adhered to the surface of all decellularised tissues after 1 week of cell culture. The developed method has the potential to become a cost-effective, one-step procedure for the decellularisation of dense tissues.

During the last decade, extensive research has been performed in the field of orthopedic medicine to develop cell-based therapies for the restoration of injured bone tissue. We previously demonstrated that human primary fetal bone cells (HFBCs) associated with porous scaffolds induced a bone formation in critical calvaria defect; however, the environmental factors regulating their behavior in culture have not been identified. HFBCs (human fetal femur,12 week development) were compared to marrow-derived human mesenchymal stem cells (HMSCs) for their capacity to proliferate and differentiate into osteoblasts under various culture conditions. When cultured in standard alphaMEM medium, PDGF and FGF-2 increased cell proliferation of both cell types. Investigation of the differentiating capacity of HFBCs and HMSCs in a normal culture medium indicated that HFBCs expressed higher expression levels of RUNX2, OSX, and osteogenic markers compared with HMSCs, while SOX9 was expressed at very low levels in both cells types. However, HMSCs, but not HFBCs enhanced osteoblastic markers in response to osteogenic factors. Surprisingly, BMP-2 with osteogenic factors increased cell numbers and reduced osteoblastic differentiation in HFBCs with the opposite effect seen in HMSCs. Associated with a higher expression of osteoblastic markers, HFBCs produced a higher calcified extra cellular matrix compared with HMSCs. Taken together, data presented in this study suggest that HFBCs have characteristics of osteoprecursor cells that are more advanced in their osteogenesis development compared with mesenchymal stem cells, making fetal cells an interesting biological tool for treatment of skeletal defects and diseases.

In this study, a new and simple glassy carbon electrode modified with carbon nanohorns (SWCNH/GCE) was used for the determination of Cr(VI) in aqueous matrices via adsorptive cathodic stripping voltammetry (AdCSV). The modified electrode was characterized via field emission scanning electron microscopy and cyclic voltammetry, which revealed a homogeneous distribution of spherical agglomerates of SWCNH on the electrode surface. The modification increased the electrochemically active area from 0.10 cm 2 ± 0.01 (GCE) to 0.16 cm 2 ± 0.01 (SWCNH/GCE). The optimized analytical conditions were as follows: a supporting electrolyte (0.15 mol L -1 HCl), an accumulation potential of 0.8 V versus Ag/AgCl, and an accumulation time of 240 s. Validation of the analytical methodology was performed, obtaining a linear range between 20 and 100 µg L -1 , a limit of detection of 3.5 µg L -1 , and a limit of quantification of 11.6 µg L -1 with good accuracy and precision. The method was applied to the analysis of spiked tap water samples, and the results were compared using a flame atomic absorption spectrophotometer (FAAS) with no significant statistical differences.

Single-cell microcultures (SCMs) form a monosynaptic circuit that allows stimulation and recording of postsynaptic responses using a single electrode. Here, we present a protocol to establish autaptic cultures from rat superior cervical ganglion neurons. We describe the steps for preparing SCMs, recording synaptic currents, and identifying and processing the recorded neurons for electron microscopy. We then detail procedures for visualizing synapses. This protocol is illustrated by correlating evoked and spontaneous neurotransmitter release with the ultrastructural features of synapses recorded. For complete details on the use and execution of this protocol, please refer to Velasco et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Urine is a popular biofluid for metabolomics studies due to its simple, non-invasive collection and its availability in large quantities, permitting frequent sampling, replicate analyses, and sample banking. The biggest disadvantage with using urine is that it exhibits significant variability in concentration and composition within an individual over relatively short periods of time (arising from various external factors and internal processes regulating the body's water and solute content). In treating the data from urinary metabolomics studies, one must account for the natural variability of urine concentrations to avoid erroneous data interpretation. Amongst various proposed approaches to account for broadly varying urine sample concentrations, normalization to creatinine has been widely accepted and is most commonly used. MS total useful signal (MSTUS) is another normalization method that has been recently reported for mass spectrometry (MS)-based metabolomics studies. Herein, we explored total useful peak area (TUPA), a modification of MSTUS that is applicable to GC×GC-TOFMS (and data from other separations platforms), for sample normalization in urinary metabolomics studies. Performance of TUPA was compared to the two most common normalization approaches, creatinine adjustment and Total Peak Area (TPA) normalization. Each normalized dataset was evaluated using Principal Component Analysis (PCA). The results showed that TUPA outperformed alternative normalization methods to overcome urine concentration variability. Results also conclusively demonstrate the risks in normalizing data to creatinine., Competing Interests: The authors declare no conflict of interest.