Advancing Tumor Microenvironment Research by Combining Organs-on-Chips and Biosensors.

Organs-on-chips are microfluidic tissue-engineered models that offer unprecedented dynamic control over cellular microenvironments, emulating key functional features of organs or tissues. Sensing technologies are increasingly becoming an essential part of such advanced model systems for real-time detection of cellular behavior and systemic-like events. The fast-developing field of organs-on-chips is accelerating the development of biosensors toward easier integration, thus smaller and less invasive, leading to enhanced access and detection of (patho-) physiological biomarkers. The outstanding combination of organs-on-chips and biosensors holds the promise to contribute to more effective treatments, and, importantly, improve the ability to detect and monitor several diseases at an earlier stage, which is particularly relevant for complex diseases such as cancer. Biosensors coupled with organs-on-chips are currently being devised not only to determine therapy effectiveness but also to identify emerging cancer biomarkers and targets. The ever-expanding use of imaging modalities for optical biosensors oriented toward on-chip applications is leading to less intrusive and more reliable detection of events both at the cellular and microenvironment levels. This chapter comprises an overview of hybrid approaches combining organs-on-chips and biosensors, focused on modeling and investigating solid tumors, and, in particular, the tumor microenvironment. Optical imaging modalities, specifically fluorescence and bioluminescence, will be also described, addressing the current limitations and future directions toward an even more seamless integration of these advanced technologies.

Mesenchymal Stem Cells Empowering Tendon Regenerative Therapies.

Tendon tissues have limited healing capacity. The incidence of tendon injuries and the unsatisfactory functional outcomes of tendon repair are driving the search for alternative therapeutic approaches envisioning tendon regeneration. Cellular therapies aim at delivering adequate, regeneration-competent cell types to the injured tendon and toward ultimately promoting its reconstruction and recovery of functionality. Mesenchymal stem cells (MSCs) either obtained from tendons or from non-tendon sources, like bone marrow (BM-MSCs) or adipose tissue (ASCs), have been receiving increasing attention over the years toward enhancing tendon healing. Evidences from in vitro and in vivo studies suggest MSCs can contribute to accelerate and improve the quality of tendon healing. Nonetheless, the exact mechanisms underlying these repair events are yet to be fully elucidated. This review provides an overview of the main challenges in the field of cell-based regenerative therapies, discussing the role of MSCs in boosting tendon regeneration, particularly through their capacity to enhance the tenogenic properties of tendon resident cells.

A Textile Platform Using Continuous Aligned and Textured Composite Microfibers to Engineer Tendon-to-Bone Interface Gradient Scaffolds.

Tendon-to-bone interfaces exhibit a hierarchical multitissue transition. To replicate the progression from mineralized to nonmineralized tissue, a novel 3D fibrous scaffold is fabricated with spatial control over mineral distribution and cellular alignment. For this purpose, wet-spun continuous microfibers are produced using polycaprolactone (PCL)/ gelatin and PCL/gelatin/hydroxyapatite nano-to-microparticles (HAp). Higher extrusion rates result in aligned PCL/gelatin microfibers while, in the case of PCL/gelatin/HAp, the presence of minerals leads to a less organized structure. Biological performance using human adipose-derived stem cells (hASCs) demonstrates that topography of PCL/gelatin microfibers can induce cytoskeleton elongation, resembling native tenogenic organization. Matrix mineralization on PCL/gelatin/HAp wet-spun composite microfibers suggest the production of an osteogenic-like matrix, without external addition of osteogenic medium supplementation. As proof of concept, a 3D gradient structure is produced by assembling PCL/gelatin and PCL/gelatin/HAp microfibers, resulting in a fibrous scaffold with a continuous topographical and compositional gradient. Overall, the feasibility of wet-spinning for the generation of continuously aligned and textured microfibers is demonsrated, which can be further assembled into more complex 3D gradient structures to mimic characteristic features of tendon-to-bone interfaces.

Crosstalk between adipose stem cells and tendon cells reveals a temporal regulation of tenogenesis by matrix deposition and remodeling.

Tendon injuries constitute an unmet clinical challenge owing to the limited intrinsic regenerative ability of this tissue. Cell-based therapies aim at improving tendon healing through the delicate orchestration of tissue rebuilding and regain of function. Hence, human adipose-derived stem cells (hASCs) have been proposed as a promising cell source for boosting tendon regeneration. In this work, we investigated the influence of hASCs on native human tendon-derived cells (hTDCs) through the establishment of a direct contact co-culture system. Results demonstrated that direct interactions between these cell types resulted in controlled proliferation and spontaneous cell elongation. ECM-related genes, particularly COL1A1 and TNC, and genes involved in ECM remodeling, such as MMP1, MMP2, MMP3, and TIMP1, were expressed in co-culture in a temporally regulated manner. In addition, deposition of collagen type I was accelerated in co-culture systems and favored over the production of collagen type III, resulting in an enhanced COL1/COL3 ratio as soon as 7 days. In conclusion, hASCs seem to be good candidates in modulating the behavior of native tendon cells, particularly through a balanced process of ECM synthesis and degradation.

Inhibition of cholinergic neurotransmission by ß3-adrenoceptors depends on adenosine release and A1-receptor activation in human and rat urinary bladders.

The direct detrusor relaxant effect of ß3-adrenoceptor agonists as a primary mechanism to improve overactive bladder symptoms has been questioned. Among other targets, activation of ß3-adrenoceptors downmodulate nerve-evoked acetylcholine (ACh) release, but there is insufficient evidence for the presence of these receptors on bladder cholinergic nerve terminals. Our hypothesis is that adenosine formed from the catabolism of cyclic AMP in the detrusor may act as a retrograde messenger via prejunctional A1 receptors to explain inhibition of cholinergic activity by ß3-adrenoceptors. Isoprenaline (1 µM) decreased [3H]ACh release from stimulated (10 Hz, 200 pulses) human (-47 ± 5%) and rat (-38 ± 1%) detrusor strips. Mirabegron (0.1 µM, -53 ± 8%) and CL316,243 (1 µM, -37 ± 7%) mimicked isoprenaline (1 µM) inhibition, and their effects were prevented by blocking ß3-adrenoceptors with L748,337 (30 nM) and SR59230A (100 nM), respectively, in human and rat detrusor. Mirabegron and isoprenaline increased extracellular adenosine in the detrusor. Blockage of A1 receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 nM) or the equilibrative nucleoside transporters (ENT) with dipyridamole (0.5 µM) prevented mirabegron and isoprenaline inhibitory effects. Dipyridamole prevented isoprenaline-induced adenosine outflow from the rat detrusor, and this effect was mimicked by the ENT1 inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 30 µM). Cystometry recordings in anesthetized rats demonstrated that SR59230A, DPCPX, dipyridamole, and NBTI reversed the decrease in the voiding frequency caused by isoprenaline (0.1-1,000 nM). Data suggest that inhibition of cholinergic neurotransmission by ß3-adrenoceptors results from adenosine release via equilibrative nucleoside transporters and prejunctional A1-receptor stimulation in human and rat urinary bladder.

Optimisation and validation of a HS-SPME-GC-IT/MS method for analysis of carbonyl volatile compounds as biomarkers in human urine: Application in a pilot study to discriminate individuals with smoking habits.

A new and simple analytical approach consisting of an automated headspace solid-phase microextraction (HS-SPME) sampler coupled to gas chromatography-ion trap/mass spectrometry detection (GC-IT/MS) with a prior derivatization step with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) was developed to detect volatile carbonyl metabolites with low molecular weights in human urine. A central composite design (CCD) was used to optimise the PFBHA concentration and extraction conditions that affect the efficiency of the SPME procedure. With a sample volume of 1 mL, optimal conditions were achieved by adding 300 mg/L of PFBHA and allowing the sample to equilibrate for 6 min at 62°C and then extracting the samples for 51 min at the same temperature, using a divinylbenzene/polydimethylsiloxane (DVB/PDMS) fibre. The method allowed the simultaneous identification and quantification of 44 carbonyl compounds consisting of aldehydes, dialdehydes, heterocyclic aldehydes and ketones. The method was validated with regards to the linearity, inter- and intra-day precision and accuracy. The detection limits ranged from 0.009 to 0.942 ng/mL, except for 4-hydroxy-2-nonenal (15 ng/mL), and the quantification limits varied from 0.029 to 1.66 ng/mL, except for butanal (2.78 ng/mL), 2-butanone (2.67 ng/mL), 4-heptanone (3.14 ng/mL) and 4-hydroxy-2-nonenal (50.0 ng/mL). The method accuracy was satisfactory, with recoveries ranging from 90 to 107%. The proof of applicability of the methodology was performed in a pilot target analysis of urine samples obtained from 18 healthy smokers and 18 healthy non-smokers (control group). Chemometric supervised analysis was performed using the volatile patterns acquired for these samples and clearly showed the potential of the volatile carbonyl profiles to discriminate urine from smoker and non-smoker subjects. 5-Methyl-2-furfural (p<0.0001), 2-methylpropanal, nonanal and 2-methylbutanal (p<0.05) were identified as potentially useful biomarkers to identify smoking habits.