Dysregulation of calcium homeostasis in cancer and its role in chemoresistance.

Globally, cancer, as a major public health concern, poses a severe threat to people's well-being. Advanced and specialized therapies can now cure the majority of people with early-stage cancer. However, emerging resistance to traditional and novel chemotherapeutic drugs remains a serious issue in clinical medicine. Chemoresistance often leads to cancer recurrence, metastasis, and increased mortality, accounting for 90% of chemotherapy failures. Thus, it is important to understand the molecular mechanisms of chemoresistance and find novel therapeutic approaches for cancer treatment. Among the several factors responsible for chemoresistance, calcium (Ca2+) dysregulation plays a significant role in cancer progression and chemoresistance. Therefore, targeting this derailed Ca2+ signalling for cancer therapy has become an emerging research area. Of note, the Ca2+ signal and its proteins are a multifaceted and potent tool by which cells achieve specific outcomes. Depending on cell survival needs, Ca2+ is either upregulated or downregulated in both chemosensitive and chemoresistant cancer cells. Consequently, the appropriate treatment should be selected based on Ca2+ signalling dysregulation. This review discusses the role of Ca2+ in cancer cells and the targeting of Ca2+ channels, pumps, and exchangers. Furthermore, we have emphasised the role of Ca2+ in chemoresistance and therapeutic strategies. In conclusion, targeting Ca2+ signalling is a multifaceted process. Methods such as site-specific drug delivery, target-based drug-designing, and targeting two or more Ca2+ proteins simultaneously may be explored; however, further clinical studies are essential to validate Ca2+ blockers' anti-cancer efficacy.

Macrophage Polarization Dynamics in Biomaterials: Implications for in Vitro Wound Healing.

The interaction between biomaterials and the immune system plays a pivotal role in determining the success or failure of implantable devices. Macrophages, as key orchestrators of immune responses, exhibit diverse reactions that influence tissue integration or lead to implant failure. This study focuses on unraveling the intricate relationship between macrophage phenotypes and biomaterials, specifically hydrogels, by employing THP-1 cells as a model. Through a comprehensive investigation using polysaccharide, polymer, and protein-based hydrogels, our research sheds light on how the properties of hydrogels influence macrophage polarization. Phenotypic observations, biochemical assays, surface marker expression, and gene expression profiles collectively demonstrate the differential macrophage polarization abilities of polysaccharide-, polymer-, and protein-based hydrogels. Moreover, our indirect coculture studies reveal that hydrogels fostering M2 polarization exhibit exceptional wound-healing capabilities. These findings highlight the crucial role of the hydrogel microenvironment in adjusting macrophage polarization, offering a fresh avenue for refining biomaterials to bolster advantageous immune responses and improve tissue integration. This research contributes valuable insights for designing biomaterials with tailored properties that can guide macrophage behavior, ultimately improving the overall success of implantable devices.

A Neutral Flavin-Triphenylamine Probe for Mitochondrial Bioimaging under Different Microenvironments.

A bioinspired design built around a neutral flavin-triphenylamine core has been investigated for selective mitochondrial bioimaging capabilities in different microenvironments. Significant advantages with respect to long-term tracking, faster internalization, penetrability within the spheroid structures, and strong emission signal under induced hypoxia conditions have been observed, which could offer an alternative to the existing mitotrackers for hypoxia-related biological events.

Selective Targeting of Lung Cancer Cells with Methylparaben-Tethered-Quinidine Cocrystals in 3D Spheroid Models.

The development and design of pharmaceutical cocrystals for various biological applications has garnered significant interest. In this study, we have established methodologies for the growth of the methylparaben-quinidine cocrystal (MP-QU), which exhibits a well-defined order that favors structure-property correlation. To confirm the cocrystal formation, we subjected the cocrystals to various physicochemical analyses such as powder X-ray diffraction (PXRD), single-crystal X-ray diffraction (SCXRD), Raman, and IR spectroscopy. The results of the XRD pattern comparisons indicated no polymorphisms, and density functional theory (DFT) studies in both gaseous and liquid phases revealed enhanced stability. Our in silico docking studies demonstrated the cocrystal's high-affinity binding towards cancer-specific epidermal growth factor receptor (EGFR), Janus kinase (JAK), and other receptors. Furthermore, in vitro testing against three-dimensional (3D) spheroids of lung cancer (A549) and normal fibroblast cells (L929) demonstrated the cocrystal's higher anticancer potential, supported by cell viability measurements and live/dead assays. Interestingly, the cocrystal showed selectivity between cancerous and normal 3D spheroids. We found that the MP-QU cocrystal inhibited migration and invadopodia formation of cancer spheroids in a favorable 3D microenvironment.

Investigation of microstructural failure in the human cornea through fracture tests.

Fracture toughness of the human cornea is one of the critical parameters in suture-involved corneal surgeries and the development of bioengineered mimetics of the human cornea. The present article systematically studied the fracture characteristics of the human cornea to evaluate its resistance to tear in the opening (Mode-I) and trouser tear mode (Mode-III). Tear experiments reveal the dependency of the fracture behavior on the notch size and its location created in the corneal specimens. The findings indicate lamellar tear and collagen fiber pull-out as a failure mechanism in trouser tear and opening mode tests, respectively. Experimental results have shown a localized variation of tear behavior in trouser tear mode and indicated an increasing resistance to tear from the corneal center to the periphery. This article demonstrated the complications of evaluating fracture toughness in opening mode and showed that the limbus was weaker than the cornea and sclera against tearing. The overall outcomes of the present study help in designing experiments to understand the toughness of the diseased tissues, understanding the effect of the suturing location and donor placement, and creating numerical models to study parameters affecting corneal replacement surgery.

On-chip mixing of cancer cells and drug using LED enabled 2D opto-wetting droplet platforms.

Droplets of microliter size serve as miniaturized reaction chambers for practical lab on a chip (LoC) applications. The transportation and coalescence of droplets are indispensable for realizing microfluidic mixing. Light can be used as an effective tool for droplet manipulation. We report a novel platform for LED-based transport and mixing of cell-encapsulated microdroplets for evaluating dose response of cancer drugs. Microcontroller enabled LEDs (Light-emitting diodes) were used to actuate droplet movement on Azobenzene coated planar silicon substrates. Droplet transport was initiated by the spatial gradient in solid-liquid interfacial tension developed through LED triggered photoisomerization of Azobenzene substrate. Detailed UV-Visible characterization of Azobenzene molecule was performed for different LED light intensities and wavelengths. A complete standalone opto-wetting toolbox was developed by integrating various components such as a microcontroller, UV LED (385 nm), blue LED (465 nm), and Azobenzene coated photoresponsive substrate. 2D transport of DI water droplets (10-30µl) along simple trajectories was demonstrated using this device. Subsequently, the proposed opto-wetting platform was used for performing drug evaluation through on-chip mixing of droplets containing cancer cells (A549-Lung cancer cells) and cancer drug (paclitaxel). Separate cell viability analysis was performed using MTT assays, where the cytocompatibility of Azobenzene and UV light (385 nm) on A549 cells were studied. The dosage response of paclitaxel drug was studied using both MTT (3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) and live-dead cell assays. The results obtained indicate the potential use of our device as a cost-effective, reliable opto-wetting microfluidic platform for drug screening experiments.

Osteomatrix as a personalized 3D tissue-specific invasion test-bed for oral carcinoma.

The clinical challenge in the successful management of oral cancer malignancy remains in the inaccuracy of detecting regional invasion potential and inefficient treatment of recurrent tumors. The presence and extent of bone invasion by the oral tumor are of critical importance as they can influence the preoperative strategy altering the prognosis outcome. Here, we are examining the patient-specific osteotropism of oral carcinoma using a bone derived extracellular matrix. The extracellular matrix (ECM) was obtained from caprine bone by a combination of demineralization, delipidation and decellularization (D3) techniques. The bone D3-derived ECM (BdECM) tissue was characterized for analyzing the effective removal of cells, minerals, and lipids with an intact structure and chemical composition. The human adipose-derived stem cells (ADSCs) on the osteomatrix (BdECM derived hydrogel) exhibited excellent cell viability and early osteogenic differentiation capacity in vitro. Furthermore, the osteomatrix polarized monocytes towards an anti-inflammatory phenotype (M2 macrophage) indicating its low immunogenicity. In the second phase of this study, we isolated and established primary cancer cell cultures from patient-derived tissue exhibiting the cancer stem cell marker phenotype (EpCAM+/CD44high/CD24-). Moreover, the presence of side population (SP) cells confirmed a contributing factor for resistance to cancer therapy. The spheroid formed from primary cells embedded in the osteomatrix was used as a test-bed to monitor the invasion profile and screening of anti-cancer drugs. Our 3D test platform captured the inter-patient heterogeneity by displaying variation in the degree of invasion and response towards tested doses of anticancer drugs. Altogether, our data emphasize the necessity of a tissue-specific in vitro preclinical model for the evaluation of oral carcinogenesis and drug sensitivity.

3D Bioprintable Hypoxia-Mimicking PEG-Based Nano Bioink for Cartilage Tissue Engineering.

As hypoxia plays a significant role in the formation and maintenance of cartilage tissue, aiming to develop native hypoxia-mimicking tissue engineering scaffolds is an efficient method to treat articular cartilage (AC) defects. Cobalt (Co) is documented for its hypoxic-inducing effects in vitro by stabilizing the hypoxia-inducible factor-1α (HIF-1α), a chief regulator of stem cell fate. Considering this, we developed a novel three-dimensional (3D) bioprintable hypoxia-mimicking nano bioink wherein cobalt nanowires (Co NWs) were incorporated into the poly(ethylene glycol) diacrylate (PEGDA) hydrogel system as a hypoxia-inducing agent and encapsulated with umbilical cord-derived mesenchymal stem cells (UMSCs). In the current study, we investigated the impact of Co NWs on the chondrogenic differentiation of UMSCs in the PEGDA hydrogel system. Herein, the hypoxia-mimicking nano bioink (PEGDA+Co NW) was rheologically optimized to bioprint geometrically stable cartilaginous constructs. The bioprinted 3D constructs were evaluated for their physicochemical characterization, swelling-degradation behavior, mechanical properties, cell proliferation, and the expression of chondrogenic markers by histological, immunofluorescence, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) methods. The results disclosed that, compared to the control (PEGDA) group, the hypoxia-mimicking nano bioink (PEGDA+Co NW) group outperformed in print fidelity and mechanical properties. Furthermore, live/dead staining, double-stranded DNA (dsDNA) content, and glycosaminoglycans (GAGs) content demonstrated that adding low amounts of Co NWs (<20 ppm) into PEGDA hydrogel system supported UMSC adhesion, proliferation, and differentiation. Histological and immunofluorescence staining of the PEGDA+Co NW bioprinted structures revealed the production of type 2 collagen (COL2) and sulfated GAGs, rendering it a feasible option for cartilage repair. It was further corroborated by a significant upregulation of the hypoxia-mediated chondrogenic and downregulation of the hypertrophic/osteogenic marker expression. In conclusion, the hypoxia-mimicking hydrogel system, including PEGDA and Co2+ ions, synergistically directs the UMSCs toward the chondrocyte lineage without using expensive growth factors and provides an alternative strategy for translational applications in the cartilage tissue engineering field.

Advancement in "Garbage In Biomaterials Out (GIBO)" concept to develop biomaterials from agricultural waste for tissue engineering and biomedical applications.

Presently on a global scale, one of the major concerns is to find effective strategies to manage the agricultural waste to protect the environment. One strategy that has been drawing attention among the researchers is the development of biocompatible materials from agricultural waste. This strategy implies successful conversion of agricultural waste products (e.g.: cellulose, eggshell etc.) into building blocks for biomaterial development. Some of these wastes contain even bioactive compounds having biomedical applications. The replacement and augmentation of human tissue with biomaterials as alternative to traditional method not only bypasses immune-rejection, donor scarcity, and maintenance; but also provides long term solution to damaged or malfunctioning organs. Biomaterials development as one of the key challenges in tissue engineering approach, resourced from natural origin imparts better biocompatibility due to closely mimicking composition with cellular microenvironment. The "Garbage In, Biomaterials Out (GIBO)" concept, not only recycles the agricultural wastes, but also adds to biomaterial raw products for further product development in tissue regeneration. This paper reviews the conversion of garbage agricultural by-products to the biocompatible materials for various biomedical applications. Graphical abstract: The agro-waste biomass processed, purified, modified, and further utilized for the fabrication of biomaterials-based support system for tissue engineering applications to grow living body parts in vitro or in vivo.

Bioengineering strategies for 3D bioprinting of tubular construct using tissue-specific decellularized extracellular matrix.

The goal of the current study is to develop an extracellular matrix bioink that could mimic the biochemical components present in natural blood vessels. Here, we have used an innovative approach to recycle the discarded varicose vein for isolation of endothelial cells and decellularization of the same sample to formulate the decellularized extracellular matrix (dECM) bioink. The shift towards dECM bioink observed as varicose vein dECM provides the tissue-specific biochemical factors that will enhance the regeneration capability. Interestingly, the encapsulated umbilical cord mesenchymal stem cells expressed the markers of vascular smooth muscle cells because of the cues present in the vein dECM. Further, in vitro immunological investigation of dECM revealed a predominant M2 polarization which could further aid in tissue remodeling. A novel approach was used to fabricate vascular construct using 3D bioprinting without secondary support. The outcomes suggest that this could be a potential approach for patient- and tissue-specific blood vessel regeneration.

Diagnostic and Therapeutic Roles of the "Omics" in Hypoxic-Ischemic Encephalopathy in Neonates.

Perinatal asphyxia and neonatal encephalopathy remain major causes of neonatal mortality, despite the improved availability of diagnostic and therapeutic tools, contributing to neurological and intellectual disabilities worldwide. An approach using a combination of clinical data, neuroimaging, and biochemical parameters is the current strategy towards the improved diagnosis and prognosis of the outcome in neonatal hypoxic-ischemic encephalopathy (HIE) using bioengineering methods. Traditional biomarkers are of little use in this multifactorial and variable phenotype-presenting clinical condition. Novel systems of biology-based "omics" approaches (genomics, transcriptome proteomics, and metabolomics) may help to identify biomarkers associated with brain and other tissue injuries, predicting the disease severity in HIE. Biomarker studies using omics technologies will likely be a key feature of future neuroprotective treatment methods and will help to assess the successful treatment and long-term efficacy of the intervention. This article reviews the roles of different omics as biomarkers of HIE and outlines the existing knowledge of our current understanding of the clinical use of different omics molecules as novel neonatal brain injury biomarkers, which may lead to improved interventions related to the diagnostic and therapeutic aspects of HIE.

Biocompatibility-on-a-chip: Characterization and evaluation of decellularized tendon extracellular matrix (tdECM) hydrogel for 3D stem cell culture in a microfluidic device.

Researchers have always tried expensive in vitro tests to show the 3D usability of dECM. The use of tissue-specific hydrogels in a microfluidic device is rarely studied. In this study, we have used ECM obtained from goat digital flexor tendons by decellularization technique. The tdECM was characterized for its structural properties using Scanning Electron Microscopy (SEM). Collagen, dsDNA, GAGs, and protein contents were quantified using spectrophotometric assays. The cell viability and proliferation of human umbilical cord-derived mesenchymal stem cells (hUMSCs) encapsulated in the tdECM hydrogel inside the microfluidic device were checked using Calcein-AM/PI. The FTIR data showed prominent peaks of the amide group, indicating the presence of collagen. The SEM data showed intact fiber morphology after the decellularization process. There was a 95 % reduction in double-stranded DNA (dsDNA) content, proving the effectiveness of the decellularization technique. There was no significant difference in the collagen content of tdECM and the GAGs were also in the acceptable range compared to the native tissue. Over 90 % cell viability in hUMSCs was observed qualitatively and quantitatively in vitro and inside a microfluidic device. In conclusion, we characterized the tdECM hydrogel and demonstrated its compatibility with the microfluidic device.

Microfluidic Biosensor-Based Devices for Rapid Diagnosis and Effective Anti-cancer Therapeutic Monitoring for Breast Cancer Metastasis.

Breast cancer with unpredictable metastatic recurrence is the leading cause of cancer-related mortality. Early cancer detection and optimized therapy are the principal determining factors for increased survival rate. Worldwide, researchers and clinicians are in search of efficient strategies for the timely management of cancer progression. Efficient preclinical models provide information on cancer initiation, malignancy progression, relapse, and drug efficacy. The distinct histopathological features and clinical heterogeneity allows no single model to mimic breast tumor. However, engineering three-dimensional (3D) in vitro models incorporating cells and biophysical cues using a combination of organoid culture, 3D printing, and microfluidic technology could recapitulate the tumor microenvironment. These models serve to be preferable predictive models bridging the translational research gap in drug development. Microfluidic device is a cost-effective advanced in vitro model for cancer research, diagnosis, and drug assay under physiologically relevant conditions. Integrating a biosensor with microfluidics allows rapid real-time analytical validation to provide highly sensitive, specific, reproducible, and reliable outcomes. In this manner, the multi-system approach in identifying biomarkers associated with cancer facilitates early detection, therapeutic window optimization, and post-treatment evaluation.This chapter showcases the advancements related to in vitro breast cancer metastasis models focusing on microfluidic devices. The chapter aims to provide an overview of microfluidic biosensor-based devices for cancer detection and high-throughput chemotherapeutic drug screening.

Modulation of 3D Printed Calcium-Deficient Apatite Constructs with Varying Mn Concentrations for Osteochondral Regeneration via Endochondral Differentiation.

Osteochondral regeneration remains a vital problem in clinical situations affecting both bone and cartilage tissues due to the low regeneration ability of cartilage tissue. Additionally, the simultaneous regeneration of bone and cartilage is difficult to attain due to their dissimilar nature. Thus, fabricating a single scaffold for both bone and cartilage regeneration remains challenging. Biomaterials are frequently employed to promote tissue restoration, but they still cannot replicate the structure of native tissue. This study aims to create a single biomaterial that could be used to regenerate both bone and cartilage. This study focuses on synthesizing calcium-deficient apatite (CDA) with the gradual addition of manganese. The phase stability and the effect of heat treatment on manganese-doped CDA were studied using X-ray diffraction (XRD) and Rietveld refinement. The obtained powders were tested for their 3-dimensional (3D) printing ability by fabricating cuboidal 3D structures. The 3D printed scaffolds were examined for external topography using field-emission scanning electron microscopy (FE-SEM) and were subjected to compression testing. In vitro biocompatibility and differentiation studies were performed to access their biocompatibility and differentiation capabilities. Reverse transcription-quantitative PCR (RT-qPCR) analysis was done to determine the gene expression of bone- and cartilage-specific markers. Mn helps in stabilizing the ß-TCP phase beyond its sintering temperature without being degraded to α-TCP. Mn addition in CDA improves the compressive strength of the fabricated scaffolds while keeping them biocompatible. The concentrations of Mn in the CDA ceramic were found to influence the differentiation behavior of MSCs in the fabricated scaffolds. Mn-doped CDA is a promising candidate to be used as a substitute material for bone, cartilage, and osteochondral defects to facilitate repair and regeneration via endochondral differentiation. 3D printing can assist in the fabrication of a multifunctional single-unit scaffold with varied Mn concentrations, which might be able to generate the two tissues in situ in an osteochondral defect.

Antagonistic interaction between TTA-A2 and paclitaxel for anti-cancer effects by complex formation with T-type calcium channel.

Studies have shown that in cancer cells, there is an increased T-type calcium channel (TTCC) expression compared to healthy cells. Therefore, the studies targeting TTCC for cancer therapy have shown many positive outcomes. Here, we have used TTA-A2- a potent TTCC inhibitor as a test drug, and paclitaxel (PTX)- a tubule-binding anti-cancer agent as a positive control. Blocking TTCC has shown to overcome resistance in cancer cells towards anti-cancer drugs by reducing calcium influx, and some studies have shown that PTX treatment also reduces the intracellular calcium signaling in cells. So, there is a possibility that PTX might be interacting with calcium channels. Since, drug-drug interaction can cause severe side-effects, or alter the actions of each other; we aim to study the interactions among TTA-A2, PTX, and TTCC. In this study, we have used computational analysis to test the binding of TTA-A2 and PTX with TTCC. To confirm the in-silico result, we further tested these drugs in a 3D spheroid model of A549, a lung adenocarcinoma cell line. The in-silico result showed that both the drugs, TTA-A2 and PTX, could interact at the same site of TTCC to form a higher stable complex as compared to the TTCC-native. The in vitro result showed the antagonistic interaction between the drugs when they are used at the same time. By using the sequential treatment, the spheroids were sensitized by TTA-A2, before treating with PTX. The result indicated that sequential treatment could help to overcome the antagonistic interaction between the two drugs. Communicated by Ramaswamy H. Sarma.

Mechanically tunable photo-cross-linkable bioinks for osteogenic differentiation of MSCs in 3D bioprinted constructs.

3D bioprinting technique renders a plausible solution to tissue engineering applications, mainly bone tissue regeneration, which could provide the microenvironment with desired physical, chemical, and mechanical properties. However, the mechanical and structural stability of current natural polymers is a critical issue in the fabrication of bone tissue-engineered scaffolds. To overcome these issues, we have developed 3D bioprintable semi-synthetic polymers derived from natural (sodium alginate, A) and synthetic (polyethylene glycol, PEG) biopolymers. In order to enhance the cross-linking properties and biocompatibility, we have functionalized these polymers with acrylate and methacrylate chemical moieties. These selected combination of natural and synthetic polymers improved the mechanical strength due to the synergistic effect of covalent as well as ionic bond formation in the hydrogel system, which is evident from the tested tensile data. Further, the feasibility of 3D bioprinting of acrylate and methacrylate functionalized PEG and hydrogels have been tested for the biocompatibility of the fabricated structures with human umbilical cord mesenchymal stem cells (UMSCs). Further, these bioprinted scaffolds were investigated for osteogenic differentiation of UMSCs in two types of culture conditions: namely, i) with osteoinduction media (with OIM), ii) without osteoinduction media (w/o OIM). We have examined the osteoinductivity of scaffolds with the activity of alkaline phosphatase (ALP) content, and significant changes in the ALP activity was observed with the stiffness of developed materials. The extent osteogenic differentiation was observed by alizarin red staining and reverse transcription PCR analysis. Elevated levels of ALP, RUNX2 and COL1 gene expression has been observed in without OIM samples on week 1 and week 3. Further, our study showed that the synthesized alginate methacrylate (AMA) without osteoinduction supplement with young's modulus of 0.34 MPa has a significant difference in ALP quantity and gene expression over the other reported literature. Thus, this work plays a pivotal role in the development of 3D bioprintable and photo-cross-linkable hydrogels in osteogenic differentiation of mesenchymal stem cells.

A novel design of microfluidic platform for metronomic combinatorial chemotherapy drug screening based on 3D tumor spheroid model.

For treating cancer at various stages, chemotherapy drugs administered in combination provide better treatment results with lower side effects compared to single-drug therapy. However, finding the potential drug combinations has been challenging due to the large numbers of possible combinations from approved drugs and the failure of in vitro 2D well plate-based cancer models. 3D spheroid-based high-throughput microfluidic platforms recapitulate some of the important features of native tumor tissue and offer a promising alternative to evaluate the combinatory effects of the drugs. This study develops a novel polydimethylsiloxane (PDMS) based microfluidic design with a dynamic environment and strategically placed U-shaped wells for testing all seven possible combinations (three single-drug treatments, three pairwise combinations, treatment with all three drugs) of three chemotherapy drugs (Paclitaxel, Vinorelbine, and Etoposide) on lung tumor spheroids. The design of U-shaped wells has been validated with computational results. Firstly, we test all combinations of drugs on the conventional well plate in static conditions with 3D tumor spheroids. Based on static drug testing results, we show a proof-of-concept by testing the most effective drug combination on the microfluidic device in a dynamic environment. The concentration of the drugs used in combination falls below the maximum tolerated dose (MTD) of the individual drugs, towards low dose metronomic (LDM) chemotherapy. LDM combinatorial chemotherapy identified in this study can potentially lower toxicity and provide better treatment results in cancer patients. The device can be further used to culture patient-specific tumor spheroids and identify synergistic drug combinations for personalized medicine.

Beneficial effects of secretome derived from mesenchymal stem cells with stigmasterol to negate IL-1ß-induced inflammation in-vitro using rat chondrocytes-OA management.

Osteoarthritis (OA) is the most prevalent joint disease predominantly characterized by inflammation which drives cartilage destruction. Mesenchymal stem cells-condition medium (MSC-CM) or the secretome is enriched with bioactive factors and possesses anti-inflammatory and regenerative effects. The present study aimed at evaluating the effects of combining MSC-conditioned medium with stigmasterol compared with the individual treatments in alleviating interleukin-1 beta (IL-1ß)-induced inflammation in rat chondrocytes. Stigmasterol is a phytosterol exhibiting anti-inflammatory effects. IL-1ß (10 ng/ml) was used to induce inflammation and mimic OA in-vitro in primary rat articular chondrocytes. The IL-1ß-stimulated chondrocytes were treated with MSC-CM, stigmasterol, and a combination of MSC-CM and stigmasterol for 24 h. Cell viability was measured using MTT assay. Protein expression of inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), collagen II (COL2A1) and matrix metalloproteinase (MMP)-13 were evaluated by immunofluorescence. Gene expression levels of MMP-3, MMP-13 and A Disintegrin-like and Metalloproteinases with Thrombospondin Motifs (ADAMTS)-5 were measured using qRT-PCR. NF-κB signaling pathway was studied using western blotting. A significant reduction in the expression of iNOS, IL-6, MMP-3, MMP-13 and ADAMTS-5, and a significant increase in COL2A1 expression was observed in the rat chondrocytes across all the treatment groups. However, the combination treatment of MSC-CM and stigmasterol remarkably reversed the IL-1ß-induced pro-inflammatory/pro-catabolic responses to near normal levels comparable to the control group. The combination treatment (MSC-CM + stigmasterol) elicited a superior anti-inflammatory/anti-catabolic effect by inhibiting the IL-1ß-induced NF-κB activation evidenced by the negligible phosphorylation of p65 and IκBα subunits, thereby emphasizing the benefit of the combination therapy over the individual treatments.

Facile Route for 3D Printing of Transparent PETg-Based Hybrid Biomicrofluidic Devices Promoting Cell Adhesion.

3D printing has emerged as a promising fabrication technique for microfluidic devices, overcoming some of the challenges associated with conventional soft lithography. Filament-based polymer extrusion (popularly known as fused deposition modeling (FDM)) is one of the most accessible 3D printing techniques available, offering a wide range of low-cost thermoplastic polymer materials for microfluidic device fabrication. However, low optical transparency is one of the significant limitations of extrusion-based microfluidic devices, rendering them unsuitable for cell culture-related biological applications. Moreover, previously reported extrusion-based devices were largely dependent on fluorescent dyes for cell imaging because of their poor transparency. First, we aim to improve the optical transparency of FDM-based microfluidic devices to enable bright-field microscopy of cells. This is achieved using (1) transparent polymer filament materials such as poly(ethylene terephthalate) glycol (PETg), (2) optimized 3D printing process parameters, and (3) a hybrid approach by integrating 3D printed microfluidic devices with cast poly(dimethylsiloxane) (PDMS) blocks. We begin by optimizing four essential 3D printing process parameters (layer height, printing speed, cooling fan speed, and extrusion flow), affecting the overall transparency of 3D printed devices. Optimized parameters produce exceptional optical transparency close to 80% in 3D printed PETg devices. Next, we demonstrate the potential of FDM-based 3D printing to fabricate transparent micromixing devices with complex planar and nonplanar channel networks. Most importantly, cells cultured on native 3D printed PETg surfaces show excellent cell attachment, spreading, and proliferation during 3 days of culture without extracellular matrix coating or surface treatment. Next, we introduce L929 cells inside hybrid PETg-PDMS biomicrofluidic devices as a proof of concept. We demonstrate that 3D printed hybrid biomicrofluidic devices promote cell adhesion, allow bright-field microscopy, and maintain high cell viability for 3 days. Finally, we demonstrate the applicability of the proposed fabrication approach for developing 3D printed microfluidic devices from other FDM-compatible transparent polymers such as polylactic acid (PLA) and poly(methyl methacrylate) (PMMA).

Human Umbilical Cord-Derived Mesenchymal Stem Cells Promote Corneal Epithelial Repair In Vitro.

Corneal injuries are among the leading causes of blindness and vision impairment. Trauma, infectious keratitis, thermal and chemical (acids and alkali burn) injuries may lead to irreversible corneal scarring, neovascularization, conjunctivalization, and limbal stem cell deficiency. Bilateral blindness constitutes 12% of total global blindness and corneal transplantation remains a stand-alone treatment modality for the majority of end-stage corneal diseases. However, global shortage of donor corneas, the potential risk of graft rejection, and severe side effects arising from long-term use of immunosuppressive medications, demands alternative therapeutic approaches. Umbilical cord-derived mesenchymal stem cells can be isolated in large numbers using a relatively less invasive procedure. However, their role in injury induced corneal repair is largely unexplored. Here, we isolated, cultured and characterized mesenchymal stem cells from human umbilical cord, and studied the expression of mesenchymal (CD73, CD90, CD105, and CD34), ocular surface and epithelial (PAX6, WNT7A, and CK-8/18) lineage markers through immunofluorescence. The cultured human limbal and corneal epithelial cells were used as controls. Scratch assay was used to study the corneal epithelial repair potential of umbilical cord-derived mesenchymal stem cells, in vitro. The in vitro cultured umbilical cord-derived mesenchymal stem cells were plastic adherent, showed trilineage differentiation and expressed: mesenchymal markers CD90, CD105, CD73; epithelial marker CK-8/18, and ocular lineage developmental markers PAX6 and WNT-7A. Our findings suggest that umbilical cord-derived mesenchymal stem cells promote repair of the injured corneal epithelium by stimulating the proliferation of corneal epithelial cells, in vitro. They may serve as a potential non-ocular source of stem cells for treating injury induced bilateral corneal diseases.