LHPP expression in triple-negative breast cancer promotes tumor growth and metastasis by modulating the tumor microenvironment.

Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic form of breast cancer that lacks an effective targeted therapy. To identify new therapeutic targets, we investigated the phosphohistidine phosphatase, LHPP, which has been implicated in the development of several types of cancer. However, the full significance of LHPP in cancer progression remains unclear due to our limited understanding of its molecular mechanism. We found that levels of the LHPP phosphohistidine phosphatase were significantly increased in human breast cancer patients compared to normal adjacent tissues, with the highest levels in the TNBC subtype. When LHPP was knocked out in the MDA-MB-231 human TNBC cell line, cell proliferation, wound healing capacity, and invasion were significantly reduced. However, LHPP knockout in TNBC cells did not affect the phosphohistidine protein levels. Interestingly, LHPP knockout in MDA-MB-231 cells delayed tumor growth and reduced metastasis when orthotopically transplanted into mouse mammary glands. To investigate LHPP's role in breast cancer progression, we used next-generation sequencing and proximity-labeling proteomics, and found that LHPP regulates gene expression in chemokine-mediated signaling and actin cytoskeleton organization. Depletion of LHPP reduced the presence of tumor-infiltrating macrophages in mouse xenografts. Our results uncover a new tumor promoter role for LHPP phosphohistidine phosphatase in TNBC and suggest that targeting LHPP phosphatase could be a potential therapeutic strategy for TNBC.

Enhanced bone regeneration in rat calvarial defects through BMP2 release from engineered poly(ethylene glycol) hydrogels.

The clinical standard therapy for large bone defects, typically addressed through autograft or allograft donor tissue, faces significant limitations. Tissue engineering offers a promising alternative strategy for the regeneration of substantial bone lesions. In this study, we harnessed poly(ethylene glycol) (PEG)-based hydrogels, optimizing critical parameters including stiffness, incorporation of arginine-glycine-aspartic acid (RGD) cell adhesion motifs, degradability, and the release of BMP2 to promote bone formation. In vitro we demonstrated that human bone marrow derived stromal cell (hBMSC) proliferation and spreading strongly correlates with hydrogel stiffness and adhesion to RGD peptide motifs. Moreover, the incorporation of the osteogenic growth factor BMP2 into the hydrogels enabled sustained release, effectively inducing bone regeneration in encapsulated progenitor cells. When used in vivo to treat calvarial defects in rats, we showed that hydrogels of low and intermediate stiffness optimally facilitated cell migration, proliferation, and differentiation promoting the efficient repair of bone defects. Our comprehensive in vitro and in vivo findings collectively suggest that the developed hydrogels hold significant promise for clinical translation for bone repair and regeneration by delivering sustained and controlled stimuli from active signaling molecules.

In Vitro and Ectopic In Vivo Studies toward the Utilization of Rapidly Isolated Human Nasal Chondrocytes for Single-Stage Arthroscopic Cartilage Regeneration Therapy.

Nasal chondrocytes (NCs) have a higher and more reproducible chondrogenic capacity than articular chondrocytes, and the engineered cartilage tissue they generate in vitro has been demonstrated to be safe in clinical applications. Here, we aimed at determining the feasibility for a single-stage application of NCs for cartilage regeneration under minimally invasive settings. In particular, we assessed whether NCs isolated using a short collagenase digestion protocol retain their potential to proliferate and chondro-differentiate within an injectable, swiftly cross-linked and matrix-metalloproteinase (MMP)-degradable polyethylene glycol (PEG) gel enriched with human platelet lysate (hPL). NC-hPL-PEG gels were additionally tested for their capacity to generate cartilage tissue in vivo and to integrate into cartilage/bone compartments of human osteochondral plugs upon ectopic subcutaneous implantation into nude mice. NCs isolated with a rapid protocol and embedded in PEG gels with hPL at low cell density were capable of efficiently proliferating and of generating tissue rich in glycosaminoglycans and collagen II. NC-hPL-PEG gels developed into hyaline-like cartilage tissues upon ectopic in vivo implantation and integrated with surrounding native cartilage and bone tissues. The delivery of NCs in PEG gels containing hPL is a feasible strategy for cartilage repair and now requires further validation in orthotopic in vivo models.

Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease-Related Cargo Absent in EVs from 2D Cultures.

Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce clinically relevant cancer phenotypes in vitro. However, their effect on molecular cargo of secreted extracellular vesicles (EVs) has not yet been investigated. Here, the impact of hydrogel-based 3D engineered microtissues on EVs secreted by benign and malignant prostate cells is assessed. Compared to 2D cultures, yield of EVs per cell is significantly increased for cancer cells cultured in 3D. Whole transcriptome sequencing and proteomics of 2D-EV and 3D-EV samples reveal stark contrasts in molecular cargo. For one cell type in particular, LNCaP, enrichment is observed exclusively in 3D-EVs of GDF15, FASN, and TOP1, known drivers of prostate cancer progression. Using imaging flow cytometry in a novel approach to validate a putative EV biomarker, colocalization in single EVs of GDF15 with CD9, a universal EV marker, is demonstrated. Finally, in functional assays it is observed that only 3D-EVs, unlike 2D-EVs, confer increased invasiveness and chemoresistance to cells in 2D. Collectively, this study highlights the value of engineered 3D microtissue cultures for the study of bona fide EV cargoes and their potential to identify biomarkers that are not detectable in EVs secreted by cells cultured in standard 2D conditions.

Smart Hydrogels for the Augmentation of Bone Regeneration by Endogenous Mesenchymal Progenitor Cell Recruitment.

The treatment of bone defects with recombinant bone morphogenetic protein-2 (BMP-2) requires high doses precluding broad clinical application. Here, a bioengineering approach is presented that strongly improves low-dose BMP-2-based bone regeneration by mobilizing healing-associated mesenchymal progenitor cells (MPCs). Smart synthetic hydrogels are used to trap and study endogenous MPCs trafficking to bone defects. Hydrogel-trapped and prospectively isolated MPCs differentiate into multiple lineages in vitro and form bone in vivo. In vitro screenings reveal that platelet-derived growth factor BB (PDGF-BB) strongly recruits prospective MPCs making it a promising candidate for the engineering of hydrogels that enrich endogenous MPCs in vivo. However, PDGF-BB inhibits BMP-2-mediated osteogenesis both in vitro and in vivo. In contrast, smart two-way dynamic release hydrogels with fast-release of PDGF-BB and sustained delivery of BMP-2 beneficially promote the healing of bone defects. Collectively, it is shown that modulating the dynamics of endogenous progenitor cells in vivo by smart synthetic hydrogels significantly improves bone healing and holds great potential for other advanced applications in regenerative medicine.

Extracellular Matrix Production by Mesenchymal Stromal Cells in Hydrogels Facilitates Cell Spreading and Is Inhibited by FGF-2.

In native tissues, the interaction between cells and the surrounding extracellular matrix (ECM) is reciprocal, as cells not only receive signals from the ECM but also actively remodel it through secretion of cell-derived ECM. However, very little is known about the reciprocal interaction between cells and their secreted ECM within synthetic biomaterials that mimic the ECM for use in engineering of tissues for regenerative medicine or as tissue models. Here, poly(ethylene glycol) (PEG) hydrogels with fully defined biomaterial properties are used to investigate the emerging role of cell-derived ECM on culture outcomes. It is shown that human mesenchymal stromal cells (MSCs) secrete ECM proteins into the pericellular space early after encapsulation and that, even in the absence of material-presented cell adhesion motifs, cell-derived fibronectin enables cell spreading. Then, it is investigated how different culture conditions influence MSC ECM expression in hydrogels. Most strikingly, it is found by RNA sequencing that the fibroblast growth factor 2 (FGF-2) changes ECM gene expression and, in particular, decreases the expression of structural ECM components including fibrillar collagens. In summary, this work shows that cell-derived ECM is a guiding cue in 3D hydrogels and that FGF-2 is a potentially important ECM regulator within bioengineered cell and tissue systems.

A Versatile Biosynthetic Hydrogel Platform for Engineering of Tissue Analogues.

For creating functional tissue analogues in tissue engineering, stem cells require very specific 3D microenvironments to thrive and mature. Demanding (stem) cell types that are used nowadays can find such an environment in a heterogeneous protein mixture with the trade name Matrigel. Several variations of synthetic hydrogel platforms composed of poly(ethylene glycol) (PEG), which are spiked with peptides, have been recently developed and shown equivalence to Matrigel for stem cell differentiation. Here a clinically relevant hydrogel platform, based on PEG and gelatin, which even outperforms Matrigel when targeting 3D prevascularized bone and liver organoid tissue engineering models is presented. The hybrid hydrogel with natural and synthetic components stimulates efficient cell differentiation, superior to Matrigel models. Furthermore, the strength of this hydrogel lies in the option to covalently incorporate unmodified proteins. These results demonstrate how a hybrid hydrogel platform with intermediate biological complexity, when compared to existing biological materials and synthetic PEG-peptide approaches, can efficiently support tissue development from human primary cells.

Expanded skeletal stem and progenitor cells promote and participate in induced bone regeneration at subcritical BMP-2 dose.

The regeneration of large bone defects remains an unsolved clinical problem, which could benefit from recent findings on the biology of skeletal stem and progenitor cells. The elucidation of conditions to specifically control their dynamic and function will likely enable the development of novel treatment strategies. In this study, we aimed at dissecting the role of osteogenic cues and skeletal stem (SSC) and progenitor cell (BCSP) recruitment during biomimetic hydrogel-assisted bone regeneration. To do so, we employed a biomimetic synthetic hydrogel based on poly (ethylene glycol) (PEG), highly controllable and enzymatically crosslinkable. We show that hydrogel-released bone morphogenetic protein-2 (BMP-2) dose-dependently promoted the enrichment of both SSCs and BCSPs within bone defects. Furthermore, we demonstrate that prospectively isolated neonatal bone-derived, as well as expanded SSCs and BCSPs, differentiate into osteogenic cells and enhance the healing of bone defects by low BMP-2 releasing biomaterials. These results indicate that growth factor releasing materials should be designed to first augment the number of SSCs and BCSPs, followed by their osteogenic differentiation to potentiate the healing of bone defects. Additionally, we demonstrate that expanded SSCs and BCSPs are easily accessible cell sources that allow the study of novel bone healing regimen under controlled in vitro and in vivo conditions.

Hyperphysiological compression of articular cartilage induces an osteoarthritic phenotype in a cartilage-on-a-chip model.

Owing to population aging, the social impact of osteoarthritis (OA)-the most common musculoskeletal disease-is expected to increase dramatically. Yet, therapy is still limited to palliative treatments or surgical intervention, and disease-modifying OA (DMOA) drugs are scarce, mainly because of the absence of relevant preclinical OA models. Therefore, in vitro models that can reliably predict the efficacy of DMOA drugs are needed. Here, we show, using a newly developed microphysiological cartilage-on-a-chip model that enables the application of strain-controlled compression to three-dimensional articular cartilage microtissue, that a 30% confined compression recapitulates the mechanical factors involved in OA pathogenesis and is sufficient to induce OA traits. Such hyperphysiological compression triggers a shift in cartilage homeostasis towards catabolism and inflammation, hypertrophy, and the acquisition of a gene expression profile akin to those seen in clinical osteoarthritic tissue. The cartilage on-a-chip model may enable the screening of DMOA candidates.

Flowable Polyethylene Glycol Hydrogels Support the in Vitro Survival and Proliferation of Dermal Progenitor Cells in a Mechanically Dependent Manner.

Cell-based therapies have garnered considerable interest largely because of their potential utility for tissue regeneration in a variety of organs, including skin. Designing vehicles that enable optimal delivery and purposeful integration of donor cells within tissues will be critical for their success. Here, we investigate the utility of an injectable, self-polymerizing, fully synthetic hydrogel in supporting the survival, proliferation, and function of cultured adult dermal progenitor cells (DPCs) which may serve as a source of renewable cells to repair severe skin injuries or restore hair growth. We show that modifying the stiffness of these transglutaminase cross-linked poly(ethylene glycol) (TG-PEG) hydrogels significantly alters DPC behavior and phenotype; increasing stiffness promotes their differentiation and migration whereas softer gels maintained them in a proliferative state. We found that 2-3% TG-PEG was optimal to promote cell expansion and survival. Unexpectedly, DPCs grown in all conditions maintained their inductive function and thus generated de novo hair follicles. Our data suggests that TG-PEG hydrogels may be a versatile platform for stem and progenitor cell transplantation and fate specification while maintaining functional competence.

Notch-inducing hydrogels reveal a perivascular switch of mesenchymal stem cell fate.

The fate of mesenchymal stem cells (MSCs) in the perivascular niche, as well as factors controlling their fate, is poorly understood. Here, we study MSCs in the perivascular microenvironment of endothelial capillaries by modifying a synthetic 3D biomimetic poly(ethylene glycol) (PEG)-hydrogel system in vitro We show that MSCs together with endothelial cells form micro-capillary networks specifically in soft PEG hydrogels. Transcriptome analysis of human MSCs isolated from engineered capillaries shows a prominent switch in extracellular matrix (ECM) production. We demonstrate that the ECM phenotypic switch of MSCs can be recapitulated in the absence of endothelial cells by functionalizing PEG hydrogels with the Notch-activator Jagged1. Moreover, transient culture of MSCs in Notch-inducing microenvironments reveals the reversibility of this ECM switch. These findings provide insight into the perivascular commitment of MSCs by use of engineered niche-mimicking synthetic hydrogels.

Spatially confined induction of endochondral ossification by functionalized hydrogels for ectopic engineering of osteochondral tissues.

Despite the various reported approaches to generate osteochondral composites by combination of different cell types and materials, engineering of templates with the capacity to autonomously and orderly develop into cartilage-bone bi-layered structures remains an open challenge. Here, we hypothesized that the embedding of cells inducible to endochondral ossification (i.e. bone marrow derived mesenchymal stromal cells, BMSCs) and of cells capable of robust and stable chondrogenesis (i.e. nasal chondrocytes, NCs) adjacent to each other in bi-layered hydrogels would develop directly in vivo into osteochondral tissues. Poly(ethylene glycol) (PEG) hydrogels were functionalized with TGFß3 or BMP-2, enzymatically polymerized encapsulating human BMSCs, combined with a hydrogel layer containing human NCs and ectopically implanted in nude mice without pre-culture. The BMSC-loaded layers reproducibly underwent endochondral ossification and generated ossicles containing bone and marrow. The NC-loaded layers formed cartilage tissues, which (under the influence of BMP-2 but not of TGFß3 from the neighbouring layer) remained phenotypically stable. The proposed strategy, resulting in orderly connected osteochondral composites, should be further assessed for the repair of osteoarticular defects and will be useful to model developmental processes leading to cartilage-bone interfaces.

Polyisocyanopeptide hydrogels: A novel thermo-responsive hydrogel supporting pre-vascularization and the development of organotypic structures.

Molecular and mechanical interactions with the 3D extracellular matrix are essential for cell functions such as survival, proliferation, migration, and differentiation. Thermo-responsive biomimetic polyisocyanopeptide (PIC) hydrogels are promising new candidates for 3D cell, tissue, and organ cultures. This is a synthetic, thermo-responsive and stress-stiffening material synthesized via polymerization of the corresponding monomers using a nickel perchlorate as a catalyst. It can be tailored to meet various demands of cells by modulating its stiffness and through the decoration of the polymer with short GRGDS peptides using copper free click chemistry. These peptides make the hydrogels biocompatible by mimicking the binding sites of certain integrins. This study focuses on the optimization of the PIC polymer properties for efficient cell, tissue and organ development. Screening for the optimal stiffness of the hydrogel and the ideal concentration of the GRGDS ligand conjugated with the polymer, enabled cell proliferation, migration and differentiation of various primary cell types of human origin. We demonstrate that fibroblasts, endothelial cells, adipose-derived stem cells and melanoma cells, do survive, thrive and differentiate in optimized PIC hydrogels. Importantly, these hydrogels support the spontaneous formation of complex structures like blood capillaries in vitro. Additionally, we utilized the thermo-responsive properties of the hydrogels for a rapid and gentle recovery of viable cells. Finally, we show that organotypic structures of human origin grown in PIC hydrogels can be successfully transplanted subcutaneously onto immune-compromised rats, on which they survive and integrate into the surrounding tissue. STATEMENT OF SIGNIFICANCE: Molecular and mechanical interactions with the surrounding environment are essential for cell functions. Although 2D culture systems greatly contributed to our understanding of complex biological phenomena, they cannot substitute for crucial interaction that take place in 3D. 3D culture systems aim to overcome limitations of the 2D cultures and answer new questions about cell functions. Thermo-responsive biomimetic polyisocyanopeptide (PIC) hydrogels are promising new candidates for 3D cell, tissue, and organ cultures. They are synthetic and can be tailor to meet certain experimental demands. Additionally, they are characterized by strain-stiffening, a feature crucial for cell behaviour, but rare in hydrogels. Their thermos-responsive properties enable quick recovery of the cells by a simple procedure of lowering the temperature.

Single cell-laden protease-sensitive microniches for long-term culture in 3D.

Single cell-laden three-dimensional (3D) microgels that can serve to mimic stem cell niches in vitro, and are therefore termed microniches, can be efficiently fabricated by droplet-based microfluidics. In this technique an aqueous polymer solution along with a highly diluted cell solution is injected into a microfluidic device to create monodisperse pre-microgel droplets that are then solidified by a polymer crosslinking reaction to obtain monodisperse single cell-laden microniches. However, problems limiting this approach studying the fate of single cells include Poisson encapsulation statistics that result in mostly empty microniches, and cells egressing from the microniches during subsequent cell culture. Here, we present a strategy to bypass Poisson encapsulation statistics in synthetic microniches by selective crosslinking of only cell-laden pre-microgel droplets. Furthermore, we show that we can position cells in the center of the microniches, and that even in protease-sensitive microniches this greatly reduces cell egress. Collectively, we present the development of a versatile protocol that allows for unprecedented efficiency in creation of synthetic protease-sensitive microniches for probing single stem cell fate in 3D.

One-Step Microfluidic Fabrication of Polyelectrolyte Microcapsules in Aqueous Conditions for Protein Release.

We report a microfluidic approach for one-step fabrication of polyelectrolyte microcapsules in aqueous conditions. Using two immiscible aqueous polymer solutions, we generate transient water-in-water-in-water double emulsion droplets and use them as templates to fabricate polyelectrolyte microcapsules. The capsule shell is formed by the complexation of oppositely charged polyelectrolytes at the immiscible interface. We find that attractive electrostatic interactions can significantly prolong the release of charged molecules. Moreover, we demonstrate the application of these microcapsules in encapsulation and release of proteins without impairing their biological activities. Our platform should benefit a wide range of applications that require encapsulation and sustained release of molecules in aqueous environments.

Dual Role of Mesenchymal Stem Cells Allows for Microvascularized Bone Tissue-Like Environments in PEG Hydrogels.

In vitro engineered tissues which recapitulate functional and morphological properties of bone marrow and bone tissue will be desirable to study bone regeneration under fully controlled conditions. Among the key players in the initial phase of bone regeneration are mesenchymal stem cells (MSCs) and endothelial cells (ECs) that are in close contact in many tissues. Additionally, the generation of tissue constructs for in vivo transplantations has included the use of ECs since insufficient vascularization is one of the bottlenecks in (bone) tissue engineering. Here, 3D cocultures of human bone marrow derived MSCs (hBM-MSCs) and human umbilical vein endothelial cells (HUVECs) in synthetic biomimetic poly(ethylene glycol) (PEG)-based matrices are directed toward vascularized bone mimicking tissue constructs. In this environment, bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2) promotes the formation of vascular networks. However, while osteogenic differentiation is achieved with BMP-2, the treatment with FGF-2 suppressed osteogenic differentiation. Thus, this study shows that cocultures of hBM-MSCs and HUVECs in biological inert PEG matrices can be directed toward bone and bone marrow-like 3D tissue constructs.

A Highly Elastic and Rapidly Crosslinkable Elastin-Like Polypeptide-Based Hydrogel for Biomedical Applications.

Elastin-like polypeptides (ELPs) are promising for biomedical applications due to their unique thermoresponsive and elastic properties. ELP-based hydrogels have been produced through chemical and enzymatic crosslinking or photocrosslinking of modified ELPs. Herein, a photocrosslinked ELP gel using only canonical amino acids is presented. The inclusion of thiols from a pair of cysteine residues in the ELP sequence allows disulfide bond formation upon exposure to UV light, leading to the formation of a highly elastic hydrogel. The physical properties of the resulting hydrogel such as mechanical properties and swelling behavior can be easily tuned by controlling ELP concentrations. The biocompatibility of the engineered ELP hydrogels is shown in vitro as well as corroborated in vivo with subcutaneous implantation of hydrogels in rats. ELP constructs demonstrate long-term structural stability in vivo, and early and progressive host integration with no immune response, suggesting their potential for supporting wound repair. Ultimately, functionalized ELPs demonstrate the ability to function as an in vivo hemostatic material over bleeding wounds.

Chondrocyte culture in three dimensional alginate sulfate hydrogels promotes proliferation while maintaining expression of chondrogenic markers.

The loss of expression of chondrogenic markers during monolayer expansion remains a stumbling block for cell-based treatment of cartilage lesions. Here, we introduce sulfated alginate hydrogels as a cartilage biomimetic biomaterial that induces cell proliferation while maintaining the chondrogenic phenotype of encapsulated chondrocytes. Hydroxyl groups of alginate were converted to sulfates by incubation with sulfur trioxide-pyridine complex (SO3/pyridine), yielding a sulfated material cross-linkable with calcium chloride. Passage 3 bovine chondrocytes were encapsulated in alginate and alginate sulfate hydrogels for up to 35 days. Cell proliferation was five-fold higher in alginate sulfate compared with alginate (p=0.038). Blocking beta1 integrins in chondrocytes within alginate sulfate hydrogels significantly inhibited proliferation (p=0.002). Sulfated alginate increased the RhoA activity of chondrocytes compared with unmodified alginate, an increase that was blocked by ß1 blocking antibodies (p=0.017). Expression and synthesis of type II collagen, type I collagen, and proteoglycan was not significantly affected by the encapsulation material evidenced by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Alginate sulfate constructs showed an opaque appearance in culture, whereas the unmodified alginate samples remained translucent. In conclusion, alginate sulfate provides a three dimensional microenvironment that promotes both chondrocyte proliferation and maintenance of the chondrogenic phenotype and represents an important advance for chondrocyte-based cartilage repair therapies providing a material in which cell expansion can be done in situ.

GFOGER-modified MMP-sensitive polyethylene glycol hydrogels induce chondrogenic differentiation of human mesenchymal stem cells.

The cellular microenvironment plays a crucial role in directing proliferation and differentiation of stem cells. Cells interact with their microenvironment via integrins that recognize certain peptide sequences of extracellular matrix proteins. This receptor-ligand binding has profound impact on cell fate. Interactions of human bone marrow mesenchymal stem cells (hMSCs) with the triple helical collagen mimetic, GPC(GPP)5-GFOGER-(GPP)5GPC-NH2, and the fibronectin adhesion peptide, RGD, were studied in degradable or nondegradable polyethylene glycol (PEG) gels formed by Michael-type addition chemistry. Proliferation, cytoskeletal morphology, and chondrogenic differentiation of encapsulated hMSCs were evaluated. The hMSCs adopted a highly spread morphology within the GFOGER-modified gels, whereas RGD induced a star-like spreading of the cells. hMSCs within GFOGER-modified degradable gels had a high proliferation rate compared with cells in peptide-free gels (p=0.017). Gene expression of type II collagen was highest in GFOGER-modified degradable gels after 21 days. Peptide incorporation increased GAG production in degradable gels after 7 and 21 days and GFOGER-modified degradable hydrogels had on average the highest GAG content, a finding that was confirmed by Alcian blue staining. In conclusion, the GFOGER peptide enhances proliferation in degradable PEG gels and provides a better chondrogenic microenvironment compared with the RGD peptide.

Reversible cation response with a protein-modified nanopipette.

The calcium ion response of a quartz nanopipette was enhanced by immobilization of calmodulin to the nanopore surface. Binding to the analyte is rapidly reversible in neutral buffer and requires no change in media or conditions to regenerate the receptor. The signal remained reproducible over numerous measurements. The modified nanopipette was used to measure binding affinity to calcium ions, with a K(d) of 6.3 ± 0.8 × 10(-5) M. This affinity is in good agreement with reported values of the solution-state protein. The behavior of such reversible nanopore-based sensors can be used to study proteins in a confined environment and may lead to new devices for continuous monitoring.