Design of a photoelectrochemical lab-on-a-chip immunosensor based on enzymatic production of quantum dots in situ.

In this work we report the development and validation of a photoelectrochemical immunosensor on the basis of alkaline phosphatase (ALP)-linked immunoassay for the detection of human serum albumin as a model analyte. In this biosensor, oriented immobilization of capture antibodies on aminated polystyrene was achieved via physical adsorption. After the interaction with the analyte, ALP immobilised on the surface through the sandwich immunoassay catalyses the hydrolysis of sodium thiophosphate (TP) to hydrogen sulphide (H2S) which in the presence of cadmium ions yields CdS quantum dots (QDs). The electrical current is generated in the course of the photoelectrochemical process (PEC) during irradiation of the CdS QDs with a UV LED (365 nm) on home-made screen-printed carbon electrodes modified with a conductive polymer. Reaction time, steps and volumes were optimized for the miniaturization of the process in order to develop a lab-on-a-chip platform. The microfluidic system was designed with optimised parameters to fabricate the immunosensor combining the immunoassay with PEC detection. The final system presents a sensitivity comparable to that of the commercial kit thanks to the signal amplification enabled by the enzymatic growth of CdS QDs in situ. This photoelectrochemical immunosensing strategy potentially opens up a new avenue for the detection of a wide range of analytes of interest due to the universal and effective enzymatic signal amplification method. Moreover, the developed bioanalytical device allows for a great reduction of time and reagents compared to exiting commercial assays, making it suitable for point-of-care applications.

Functionality of macrophages encapsulated in porcine decellularized adipose matrix hydrogels and interaction with Candida albicans.

Extracellular matrix hydrogels are considered one of the most suitable biomaterials for tissue regeneration due to their similarity with the extracellular microenvironment of the native tissue. Their properties are dependent on their composition, material concentration, fiber density and the fabrication approaches, among other factors. The encapsulation of immune cells in this kind of hydrogels, both in absence or presence of a pathogen, represents a promising strategy for the development of platforms that mimic healthy and infected tissues, respectively. In this work, we have encapsulated macrophages in 3D hydrogels of porcine decellularized adipose matrices (pDAMs) without and with the Candida albicans fungus, as 3D experimental models to study the macrophage immunocompetence in a closer situation to the physiological conditions and to mimic an infection scenario. Our results indicate that encapsulated macrophages preserve their functionality within these pDAM hydrogels and phagocytose live pathogens. In addition, their behavior is influenced by the hydrogel pore size, inversely related to the hydrogel concentration. Thus, larger pore size promotes the polarization of macrophages towards M2 phenotype along the time and enhances their phagocytosis capability. It is important to point out that encapsulated macrophages in absence of pathogen showed an M2 phenotype, but macrophages coencapsulated with C. albicans can switch towards an M1 inflammatory phenotype to resolve the infection, depending on the fungus quantity. The present study reveals that pDAM hydrogels preserve the macrophage plasticity, demonstrating their relevance as new models for macrophage-pathogen interaction studies that mimic an infection scenario with application in regenerative medicine research.

Biocompatible adipose extracellular matrix and reduced graphene oxide nanocomposite for tissue engineering applications.

Despite the immense need for effective treatment of spinal cord injury (SCI), no successful repair strategy has yet been clinically implemented. Multifunctional biomaterials, based on porcine adipose tissue-derived extracellular matrix (adECM) and reduced graphene oxide (rGO), were recently shown to stimulate in vitro neural stem cell growth and differentiation. Nevertheless, their functional performance in clinically more relevant in vivo conditions remains largely unknown. Before clinical application of these adECM-rGO nanocomposites can be considered, a rigorous assessment of the cytotoxicity and biocompatibility of these biomaterials is required. For instance, xenogeneic adECM scaffolds could still harbour potential immunogenicity following decellularization. In addition, the toxicity of rGO has been studied before, yet often in experimental settings that do not bear relevance to regenerative medicine. Therefore, the present study aimed to assess both the in vitro as well as in vivo safety of adECM and adECM-rGO scaffolds. First, pulmonary, renal and hepato-cytotoxicity as well as macrophage polarization studies showed that scaffolds were benign invitro. Then, a laminectomy was performed at the 10th thoracic vertebra, and scaffolds were implanted directly contacting the spinal cord. For a total duration of 6 weeks, animal welfare was not negatively affected. Histological analysis demonstrated the degradation of adECM scaffolds and subsequent tissue remodeling. Graphene-based scaffolds showed a very limited fibrous encapsulation, while rGO sheets were engulfed by foreign body giant cells. Furthermore, all scaffolds were infiltrated by macrophages, which were largely polarized towards a pro-regenerative phenotype. Lastly, organ-specific histopathology and biochemical analysis of blood did not reveal any adverse effects. In summary, both adECM and adECM-rGO implants were biocompatible upon laminectomy while establishing a pro-regenerative microenvironment, which justifies further research on their therapeutic potential for treatment of SCI.

Interfacing reduced graphene oxide with an adipose-derived extracellular matrix as a regulating milieu for neural tissue engineering.

Enthralling evidence of the potential of graphene-based materials for neural tissue engineering is motivating the development of scaffolds using various structures related to graphene such as graphene oxide (GO) or its reduced form. Here, we investigated a strategy based on reduced graphene oxide (rGO) combined with a decellularized extracellular matrix from adipose tissue (adECM), which is still unexplored for neural repair and regeneration. Scaffolds containing up to 50 wt% rGO relative to adECM were prepared by thermally induced phase separation assisted by carbodiimide (EDC) crosslinking. Using partially reduced GO enables fine-tuning of the structural interaction between rGO and adECM. As the concentration of rGO increased, non-covalent bonding gradually prevailed over EDC-induced covalent conjugation with the adECM. Edge-to-edge aggregation of rGO favours adECM to act as a biomolecular physical crosslinker to rGO, leading to the softening of the scaffolds. The unique biochemistry of adECM allows neural stem cells to adhere and grow. Importantly, high rGO concentrations directly control cell fate by inducing the differentiation of both NE-4C cells and embryonic neural progenitor cells into neurons. Furthermore, primary astrocyte fate is also modulated as increasing rGO boosts the expression of reactivity markers while unaltering the expression of scar-forming ones.

Enhanced Adipogenic Differentiation of Human Dental Pulp Stem Cells in Enzymatically Decellularized Adipose Tissue Solid Foams.

Engineered 3D human adipose tissue models and the development of physiological human 3D in vitro models to test new therapeutic compounds and advance in the study of pathophysiological mechanisms of disease is still technically challenging and expensive. To reduce costs and develop new technologies to study human adipogenesis and stem cell differentiation in a controlled in vitro system, here we report the design, characterization, and validation of extracellular matrix (ECM)-based materials of decellularized human adipose tissue (hDAT) or bovine collagen-I (bCOL-I) for 3D adipogenic stem cell culture. We aimed at recapitulating the dynamics, composition, and structure of the native ECM to optimize the adipogenic differentiation of human mesenchymal stem cells. hDAT was obtained by a two-enzymatic step decellularization protocol and post-processed by freeze-drying to produce 3D solid foams. These solid foams were employed either as pure hDAT, or combined with bCOL-I in a 3:1 proportion, to recreate a microenvironment compatible with stem cell survival and differentiation. We sought to investigate the effect of the adipogenic inductive extracellular 3D-microenvironment on human multipotent dental pulp stem cells (hDPSCs). We found that solid foams supported hDPSC viability and proliferation. Incubation of hDPSCs with adipogenic medium in hDAT-based solid foams increased the expression of mature adipocyte LPL and c/EBP gene markers as determined by RT-qPCR, with respect to bCOL-I solid foams. Moreover, hDPSC capability to differentiate towards adipocytes was assessed by PPAR-γ immunostaining and Oil-red lipid droplet staining. We found out that both hDAT and mixed 3:1 hDAT-COL-I solid foams could support adipogenesis in 3D-hDPSC stem cell cultures significantly more efficiently than solid foams of bCOL-I, opening the possibility to obtain hDAT-based solid foams with customized properties. The combination of human-derived ECM biomaterials with synthetic proteins can, thus, be envisaged to reduce fabrication costs, thus facilitating the widespread use of autologous stem cells and biomaterials for personalized medicine.