Integrated grade-wise profiling analysis reveals potential plasma miR-373-3p as prognostic indicator in Prostate Cancer & its target KPNA2.

Background: Plasma microRNAs (miRNAs) have recently garnered attention for their potential as stable biomarkers in the context of Prostate Cancer (PCa), demonstrating established associations with tumor grade, biochemical recurrence (BCR), and metastasis. This study seeks to assess the utility of plasma miRNAs as prognostic indicators for distinguishing between high-grade and low-grade PCa, and to explore their involvement in PCa pathogenesis. Methodology: We conducted miRNA profiling in both plasma and tissue specimens from patients with varying PCa grades. Subsequently, the identified miRNAs were validated in a substantial independent PCa cohort. Furthermore, we identified and confirmed the gene targets of these selected miRNAs through Western blot analysis. Results: In our plasma profiling investigation, we identified 98, 132, and 154 differentially expressed miRNAs (DEMs) in high-grade PCa vs. benign prostatic hyperplasia (BPH), low-grade PCa vs. BPH, and high-grade PCa vs. low-grade PCa, respectively. Our tissue profiling study revealed 111, 132, and 257 statistically significant DEMs for the same comparisons. Notably, miR-373-3p emerged as the sole consistently dysregulated miRNA in both plasma and tissue samples of PCa. This miRNA displayed significant overexpression in plasma and tissue samples, with fold changes of 3.584 ± 0.5638 and 8.796 ± 1.245, respectively. Furthermore, we observed a significant reduction in KPNA2 protein expression in PCa. Conclusion: Our findings lend support to the potential of plasma miR-373-3p as a valuable biomarker for predicting and diagnosing PCa. Additionally, this miRNA may contribute to the progression of PCa by inhibiting KPNA2 expression, shedding light on its role in the disease.

Recessive variants in the intergenic NOS1AP-C1orf226 locus cause monogenic kidney disease responsive to anti-proteinuric treatment.

In genetic disease, an accurate expression landscape of disease genes and faithful animal models will enable precise genetic diagnoses and therapeutic discoveries, respectively. We previously discovered that variants in NOS1AP , encoding nitric oxide synthase 1 (NOS1) adaptor protein, cause monogenic nephrotic syndrome (NS). Here, we determined that an intergenic splice product of N OS1AP / Nos1ap and neighboring C1orf226/Gm7694 , which precludes NOS1 binding, is the predominant isoform in mammalian kidney transcriptional and proteomic data. Gm7694 -/- mice, whose allele exclusively disrupts the intergenic product, developed NS phenotypes. In two human NS subjects, we identified causative NOS1AP splice variants, including one predicted to abrogate intergenic splicing but initially misclassified as benign based on the canonical transcript. Finally, by modifying genetic background, we generated a faithful mouse model of NOS1AP -associated NS, which responded to anti-proteinuric treatment. This study highlights the importance of intergenic splicing and a potential treatment avenue in a mendelian disorder.

Prenatal arsenic exposure alters keratinocyte stem cell fate through persistent activation of IGF2R-MAPK cascade leading to aggravated skin carcinogenesis in mice offspring.

Chronic exposure to arsenic (As) promotes skin carcinogenesis in humans and potentially disturbs resident stem cell dynamics, particularly during maternal and early life exposure. In the present study, we demonstrate how only prenatal arsenic exposure disturbs keratinocyte stem cell (KSC) conditioning using a BALB/c mice model. Prenatal As exposure alters the normal stemness (CD34, KRT5), differentiation (Involucrin), and proliferation (PCNA) program in skin of offspring with progression of age as observed at 2, 10, and 18 weeks. Primary KSCs isolated from exposed animal at Day-2 showed increased survival (Bax:Bcl-xL, TUNEL assay), proliferation (BrdU), and differentiation (KRT5, Involucrin) potential through the activation of pro-carcinogenic IGF2R-MAPK cascade (IGF2R-G(α)q-MEK1-ERK1/2). This was associated with reduced enrichment of histone H3K27me3 and its methylase, EZH2 along with increased binding of demethylase, KDM6A at Igf2r promoter. Altered KSCs conditioning through disturbed Igf2r imprint contributed to impaired proliferation and differentiation and an aggravated tumor response in offspring.

Inhibition of autocrine HGF maturation overcomes cetuximab resistance in colorectal cancer.

Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC.

Emerging role of miRNA in prostate cancer: A future era of diagnostic and therapeutics.

Prostate cancer (PCa) is the most common cancer in men (20%) and is responsible for 6.8% (1/5) of all cancer-related deaths in men around the world. The development and spread of prostate cancer are driven by a wide variety of genomic changes and extensive epigenetic events. Because of this, the MicroRNA (miRNA) and associated molecular mechanisms involved in PCa genesis and aggressive were only partially identified until today. The miRNAs are a newly discovered category of regulatorsthat have recently been recognized to have a significant role in regulating numerous elements of cancer mechanisms, such as proliferation, differentiation, metabolism, and apoptosis. The miRNAs are a type of small (22-24 nucleotides), non-coding, endogenous, single-stranded RNA and work as potent gene regulators. Various types of cancer, including PCa, have found evidence that miRNA genes, which are often located in cancer-related genetic regions or fragile locations, have a role in the primary steps of tumorigenesis, either as oncogenes or tumorsuppressors. To explain the link between miRNAs and their function in the initiation and advancement of PCa, we conducted a preliminary assessment. The purpose of this research was to enhance our understanding of the connection between miRNA expression profiles and PCa by elucidating the fundamental processes of miRNA expression and the target genes.

Fabrication, characterization and in vivo assessment of cardiogel loaded chitosan patch for myocardial regeneration.

Cell therapy is one of the promising approaches for cardiac repair, subsequently after infarction or injury. However, contemporary mesenchymal stromal/stem cell (MSCs) delivery strategies result in low retention and poor engraftment of donor cells, thus limiting the therapeutic efficacy. Here, we developed an engineered biomimetic cardiogel patch (EBCP) comprising of the native decellularized cardiac extracellular matrix (ECM) "cardiogel" and chitosan, leading to the efficient regeneration of injured myocardium. We also developed novel bio-adhesive that is capable of suture-free epicardial placement of EBCP to injured myocardium. We have illustrated the potential of the mussels-inspired bioadhesive system, which comprises gelatin catechol and partially oxidized chitosan, which relies on self-crosslinking capability, to promote wet adhesion. In vitro studies with isolated cardiogel promoted cell proliferation, adhesion, and migration while aiding cardiomyogenic differentiation. The EBCP's ability to protect cells from abrasion due to surrounding tissues in the myocardial infarction (MI) rat model makes it more desirable. Furthermore, the epicardial implantation of the EBCP loaded with MSCs improves the initial retention of cells and subsequent functional cardiac recovery with enhanced myocardial tissue restoration. Histological examination showed the presence of EBCP and infiltration of cells to the infarcted heart tissue. The fast and facile synthesis of bioadhesive and major therapeutic benefits of EBCP make it a potential candidate for recuperating the ailing heart.

Fluid shear stress in a logarithmic microfluidic device enhances cancer cell stemness marker expression.

Fluid shear stress (FSS) is crucial in cancer cell survival and tumor development. Noteworthily, cancer cells are exposed to several degrees of FSS in the tumor microenvironment and during metastasis. Consequently, the stemness marker expression in cancer cells changes with the FSS signal, although it is unclear how it varies with different magnitudes and during metastasis. The current work explores the stemness and drug resistance characteristics of the cervical cancer cell line HeLa in a microfluidic device with a wide range of physiological FSS. Hence, the microfluidic device was designed to achieve a logarithmic flow distribution in four culture chambers, realizing four orders of biological shear stress on a single chip. The cell cycle analysis demonstrated altered cell proliferation and mitotic arrest after FSS treatment. In addition, EdU staining revealed increased cell proliferation with medium to low FSS, whereas high shear had a suppressing effect. FSS increased competence to withstand higher intracellular ROS and mitochondrial membrane potential in HeLa. Furthermore, stemness-related gene (Sox2, N-cadherin) and cell surface marker (CD44, CD33, CD117) expressions were enhanced by FSS mechanotransduction in a magnitude-dependent manner. In summary, these stemness-like properties were concurrent with the drug resistance capability of HeLa towards doxorubicin. Overall, our microfluidic device elucidates cancer cell survival and drug resistance mechanisms during metastasis and in cancer relapse patients.

Prenatal exposure to arsenic promotes sterile inflammation through the Polycomb repressive element EZH2 and accelerates skin tumorigenesis in mouse.

Prenatal and postnatal life stress could be a potent programmer of phenotype or disease state of an individual in the later life. Prenatal arsenic exposure has been shown to promote developmental defects, low birth weight, immunotoxicity and is associated with various cancers including skin cancer in adulthood. To investigate the association between prenatal arsenic exposure and adult life skin carcinogenesis, we used a two-stage cutaneous carcinogenesis model in which BALB/c mice were prenatally exposed to 0.04 mg/kg and 0.4 mg/kg arsenic (As). Exposure to arsenic was sufficient to shorten the tumor latency period and promote epidermal hyperplasia in the offspring upon challenge with dimethylbenz[a]/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA). The levels of inflammatory and tissue microenvironment remodeling factors such as IL-1α and TNF-α were persistently elevated in the skin, and their inhibition through diacerein led to a significant decrease in the tumor response, suggesting their role in tumorigenesis. While there was overexpression of multiple epigenetic regulators at tissue level, we found decreased enrichment of Polycomb repressive complex 2 (PRC2) member EZH2 and H3K27me3 mark at the upstream of the affected inflammatory genes. The higher expression of the inflammatory genes suggests the gene specific selective nature of EZH2 repression which was also associated with increased binding of the activator KDM6a (demethylase). Further, arsenic conditioned basal keratinocytes cells (BKCs) showed increased migration and proliferation along with higher expression of tumor associated cytokines. Inhibition of EZH2 in the BKCs lead to their further upregulation suggesting that BKCs might be the potential cell type for the interaction of EZH2 and inflammatory cytokines. The present study provides new evidence for the role of PRC2 group regulators in inflammatory conditioning and development of skin cancer in offspring prenatally exposed to arsenic.

TGF-ß/Smad signaling pathway plays a crucial role in patulin-induced pro-fibrotic changes in rat kidney via modulation of slug and snail expression.

Patulin (PAT) is a mycotoxin that contaminates a variety of food and foodstuffs. Earlier in vitro and in vivo findings have indicated that kidney is one of the target organs for PAT-induced toxicity. However, no study has evaluated the chronic effects of PAT exposure at environmentally relevant doses or elucidated the detailed mechanism(s) involved. Here, using in vitro and in vivo experimental approaches, we delineated the mechanism/s involved in pro-fibrotic changes in the kidney after low-dose chronic exposure to PAT. We found that non-toxic concentrations (50 nM and 100 nM) of PAT to normal rat kidney cells (NRK52E) caused a higher generation of reactive oxygen species (ROS) (mainly hydroxyl (•OH), peroxynitrite (ONOO-), and hypochlorite radical (ClO-). PAT exposure caused the activation of mitogen-activated protein kinases (MAPKs) and its downstream c-Jun/Fos signaling pathways. Moreover, our chromatin immunoprecipitation (ChIP) analysis suggested that c-Jun/Fos binds to the promoter region of Transforming growth factor beta (TGF-ß1) and possibly induces its expression. Results showed that PAT-induced TGF-ß1 further activates the TGF-ß1/smad signaling pathways. Higher activation of slug and snail transcription factors further modulates the regulation of pro-fibrotic molecules. Similarly, in vivo results showed that PAT exposure to rats through gavage at 25 and 100 µg/kg b. wt had higher levels of kidney injury/toxicity markers namely vascular endothelial growth factor (VEGF), kidney Injury Molecule-1 (Kim-1), tissue inhibitor of metalloproteinase-1 (Timp-1), and clusterin (CLU). Additionally, histopathological analysis indicated significant alterations in renal tubules and glomeruli along with collagen deposition in PAT-treated rat kidneys. Overall, our data provide evidence of the involvement of ROS mediated MAPKs and TGF-ß1/smad pathways in PAT-induced pro-fibrotic changes in the kidney via modulation of slug and snail expression.

Genome-wide methylome pattern predictive network analysis reveal mesenchymal stem cell's propensity to undergo cardiovascular lineage.

Mesenchymal stem cells (MSCs) differentiation toward cardiovascular lineage prediction using the global methylome profile will highlight its prospective utility in regenerative medicine. We examined the propensity prediction to cardiovascular lineage using 5-Aza, a well-known cardiac lineage inducer. The customized 180 K microarray was performed and further analysis of global differentially methylated regions by Ingenuity pathway analysis (IPA) in both MSCs and 5-AC-treated MSCs. The cluster enrichment tools sorted differentially enriched genes and further annotated to construct the interactive networks. Prediction analysis revealed pathways pertaining to the cardiovascular lineage found active in the native MSCs, suggesting its higher propensity to undergo cardiac, smooth muscle cell, and endothelial lineages in vitro. Interestingly, gene interaction network also proposed majorly stemness gene network NANOG and KLF6, cardiac-specific transcription factors GATA4, NKX2.5, and TBX5 were upregulated in the native MSCs. Furthermore, the expression of cardiovascular lineage specific markers such as Brachury, CD105, CD90, CD31, KDR and various forms of ACTIN (cardiac, sarcomeric, smooth muscle) were validated in native MSCs using real time PCR and immunostaining and blotting analysis. In 5-AC-treated MSCs, mosaic interactive networks were observed to persuade towards osteogenesis and cardiac lineage, indicating that 5-AC treatment resulted in nonspecific lineage induction in MSCs, while MSCs by default have a higher propensity to undergo cardiovascular lineage. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03058-2.

Prenatal arsenic exposure induces immunometabolic alteration and renal injury in rats.

Arsenic (As) exposure is progressively associated with chronic kidney disease (CKD), a leading public health concern present worldwide. The adverse effect of As exposure on the kidneys of people living in As endemic areas have not been extensively studied. Furthermore, the impact of only prenatal exposure to As on the progression of CKD also has not been fully characterized. In the present study, we examined the effect of prenatal exposure to low doses of As 0.04 and 0.4 mg/kg body weight (0.04 and 0.4 ppm, respectively) on the progression of CKD in male offspring using a Wistar rat model. Interestingly, only prenatal As exposure was sufficient to elevate the expression of profibrotic (TGF-ß1) and proinflammatory (IL-1α, MIP-2α, RANTES, and TNF-α) cytokines at 2-day, 12- and 38-week time points in the exposed progeny. Further, alteration in adipogenic factors (ghrelin, leptin, and glucagon) was also observed in 12- and 38-week old male offspring prenatally exposed to As. An altered level of these factors coincides with impaired glucose metabolism and homeostasis accompanied by progressive kidney damage. We observed a significant increase in the deposition of extracellular matrix components and glomerular and tubular damage in the kidneys of 38-week-old male offspring prenatally exposed to As. Furthermore, the overexpression of TGF-ß1 in kidneys corresponds with hypermethylation of the TGF-ß1 gene-body, indicating a possible involvement of prenatal As exposure-driven epigenetic modulations of TGF-ß1 expression. Our study provides evidence that prenatal As exposure to males can adversely affect the immunometabolism of offspring which can promote kidney damage later in life.

Recent Advances in Cardiac Tissue Engineering for the Management of Myocardium Infarction.

Myocardium Infarction (MI) is one of the foremost cardiovascular diseases (CVDs) causing death worldwide, and its case numbers are expected to continuously increase in the coming years. Pharmacological interventions have not been at the forefront in ameliorating MI-related morbidity and mortality. Stem cell-based tissue engineering approaches have been extensively explored for their regenerative potential in the infarcted myocardium. Recent studies on microfluidic devices employing stem cells under laboratory set-up have revealed meticulous events pertaining to the pathophysiology of MI occurring at the infarcted site. This discovery also underpins the appropriate conditions in the niche for differentiating stem cells into mature cardiomyocyte-like cells and leads to engineering of the scaffold via mimicking of native cardiac physiological conditions. However, the mode of stem cell-loaded engineered scaffolds delivered to the site of infarction is still a challenging mission, and yet to be translated to the clinical setting. In this review, we have elucidated the various strategies developed using a hydrogel-based system both as encapsulated stem cells and as biocompatible patches loaded with cells and applied at the site of infarction.

Recent trends in biodegradable polyester nanomaterials for cancer therapy.

Biodegradable polyester nanomaterials-based drug delivery vehicles (DDVs) have been largely used in most of the cancer treatments due to its high biological performance and wider applications. In several previous studies, various biodegradable and biocompatible polyester backbones were used which are poly(lactic acid) (PLA), poly(ε-caprolactone) (PCL), poly(propylene fumarate) (PPF), poly(lactic-co-glycolic acid) (PLGA), poly(propylene carbonate) (PPC), polyhydroxyalkanoates (PHA), and poly(butylene succinate) (PBS). These polyesters were fabricated into therapeutic nanoparticles that carry drug molecules to the target site during the cancer disease treatment. In this review, we elaborately discussed the chemical synthesis of different synthetic polyesters and their use as nanodrug carriers (NCs) in cancer treatment. Further, we highlighted in brief the recent developments of metal-free semi-aromatic polyester nanomaterials along with its role as cancer drug delivery vehicles.

Injectable hydrogel for co-delivery of 5-azacytidine in zein protein nanoparticles with stem cells for cardiac function restoration.

Heart failure is major cause of mortality associated with mostly Myocardial infarction (MI). Transplanting mesenchymal stem cells (MSC) have exhibited potential role in myocardial regeneration. Secretion of immune-modulatory cytokines and various growth factors after transplantation plays significant role in remodelling process of MI region. However, low retention, higher shear stress during administration and rejection at host infarct environment hinders therapeutic efficacy. Myocardial regeneration demands for accurate spatio-temporal delivery of MSCs with supportive vascular network that leads to improvement of cardiac function. In this study, injectable alginate based microporous hydrogel has been used to deliver 5-Azacytidine (5-Aza) in zein protein nanoparticle with MSCs for attenuating adverse cardiac remodelling after MI. Zein nanoparticles loaded with 5-Aza were prepared by liquid-liquid dispersion, and it was found that 35% of drug was released in 7 days supported with mathematical modelling. The presence of 5-Aza and zein in developed hydrogel supported in vitro MSC proliferation, migration and angiogenesis. Significant increased expression of cardiac specific markers, GATA4, MEF2C, MLC, SERCA and NKX2.5 was observed in vitro. 5-Aza loaded protein nanoparticle with MSCs encapsulated hydrogels in rat MI model also exhibited substantial improvement of functional cardiac parameters such as cardiac output and ejection fraction. Histopathological analysis showed reduced fibrosis, attenuated infarct expansion and cardiac tissue restoration and angiogenesis. In brief, we developed nanocarrier-hydrogel system a promising strategy for co-delivering 5-Aza as cardiac differentiation cue with MSCs to achieve higher cell retention and enhanced improvement in myocardial regeneration after MI.

Perinatal exposure to silver nanoparticles reprograms immunometabolism and promotes pancreatic beta-cell death and kidney damage in mice.

Silver nanoparticles (AgNPs) are extensively utilized in food, cosmetics, and healthcare products. Though the effects of AgNPs exposure on adults are well documented, the long-term effects of gestational/perinatal exposure upon the health of offspring have not been addressed. Herein, we show that only perinatal exposure to AgNPs through the mother could lead to chronic inflammation in offspring which persists till adulthood. Further, AgNPs exposure altered offspring's immune responses against environmental stresses. AgNPs exposed offspring showed an altered response in splenocyte proliferation assay when challenged to lipopolysaccharide, concanavalin-A, AgNPs, or silver ions. Perinatal AgNPs exposure affected metabolic parameters (resistin, glucagon-like peptide-1, leptin, insulin) and upregulated JNK/P38/ERK signaling in the pancreas. We observed pancreatic damage, reduced insulin level, and increased blood glucose levels. Further, we observed renal damage, particularly to tubular and glomerular regions as indicated by histopathology and electron microscopy. Our study thus shows that only perinatal exposure to AgNPs could induce persistent inflammation, alter immune responses against foreign antigens and metabolism which may contribute to pancreatic and renal damage later in life.

Impact of National Health Mission on infant mortality in India: An interrupted time series analysis.

Government of India introduced National Rural Health Mission in 2005-now transformed into National Health Mission (NHM), to bring about architectural reforms in health sector. In this study, we evaluate the overall impact of NHM on infant mortality at national and state level. Annual data on infant mortality rate (IMR) from 1990 to 2016 were obtained from Sample Registration System bulletins. With reporting year 2009 considered as cut-off point, a two-step segmented time series regression analysis was conducted. Estimates of pre-slope, post-slope and change at the point of intervention were computed by applying auto-regressive integrated moving average (1, 0, 0) while adjusting for trend and auto correlation. We found that while IMR reduced from around 80 to 34 per 1000 live births at the national level from 1990 to 2016, the annual rate of reduction increased from 1.6 per 1000 live births before NHM to 2.2 per 1000 live births after NHM. This is estimated to have averted 248,212 infant deaths in India, between 2005 and 2017. The rate of decline in IMR accelerated in 13 out of 17 larger states, most significantly in Andhra Pradesh, Gujarat, Assam, Haryana, Punjab and Uttar Pradesh. NHM has thus been successful in accelerating the overall rate of reduction in IMR in India. There is still a need to identify the determinants of variations at state level. We recommend strengthening of NHM in terms of funding and implementation.

Functional impact of allelic variations/haplotypes of TNF-α on reproductive tract infections in Indian women.

The aim of the present study is to investigate the functional role of TNF-α single-nucleotide polymorphisms/haplotypes in an association with reproductive tract infections (RTIs) in symptomatic and asymptomatic women. A total of 850 consecutive subjects consisting of 400 cases and 450 healthy controls, were screened for RTIs, along with their risk factors and associated symptoms. The propensity score matching was performed to reduce the confounding bias arise owing to covariates and to balance the data between two groups. A total of 211 pairs (1:1) have been created. Genotyping of rs1800629 (-308) and rs361525 (-238) SNPs of TNF-α was done by PCR-RFLP followed by sequencing. The functional implication of TNF-α SNPs in an association with RTIs was also checked by using ELISA. The frequency of -238A allele and -308A allele was found to be twofold (P < 0.0001) and threefold (P < 0.0001) higher in the presence of RTIs. AA haplotype emerged as a major player in an association with RTIs and elevated TNF-α expression. The present study revealed the functional role of rs1800629 (-308) and rs361525 (-238) of TNF-α in an association with RTIs. This information may be used to establish biomarkers for an inflammatory response during the persistence of RTIs.

Developmental Exposure to Endocrine Disrupting Chemicals and Its Impact on Cardio-Metabolic-Renal Health.

Developmental origin of health and disease postulates that the footprints of early life exposure are followed as an endowment of risk for adult diseases. Epidemiological and experimental evidence suggest that an adverse fetal environment can affect the health of offspring throughout their lifetime. Exposure to endocrine disrupting chemicals (EDCs) during fetal development can affect the hormone system homeostasis, resulting in a broad spectrum of adverse health outcomes. In the present review, we have described the effect of prenatal EDCs exposure on cardio-metabolic-renal health, using the available epidemiological and experimental evidence. We also discuss the potential mechanisms of their action, which include epigenetic changes, hormonal imprinting, loss of energy homeostasis, and metabolic perturbations. The effect of prenatal EDCs exposure on cardio-metabolic-renal health, which is a complex condition of an altered biological landscape, can be further examined in the case of other environmental stressors with a similar mode of action.

Low intermittent flow promotes rat mesenchymal stem cell differentiation in logarithmic fluid shear device.

Bone marrow mesenchymal stem cells are an ideal candidate for bone tissue engineering due to their osteogenic potential. Along with chemical, mechanical signals such as fluid shear stress have been found to influence their differentiation characteristics. But the range of fluid shear experienced in vivo is too wide and difficult to generate in a single device. We have designed a microfluidic device that could generate four orders of shear stresses on adherent cells. This was achieved using a unique hydraulic resistance combination and linear optimization to the lesser total length of the circuit, making the device compact and yet generating four logarithmically increasing shear stresses. Numerical simulation depicts that, at an inlet velocity of 160 µl/min, our device generated shear stresses from 1.03 Pa to 1.09 mPa. In this condition, we successfully cultured primary rat bone marrow mesenchymal stem cells (rBMSCs) in the device for a prolonged period of time in the incubator environment (four days). Higher cell proliferation rate was observed in the intermittent flow at 1.09 mPa. At 10 mPa, both upregulation of osteogenic genes and higher alkaline phosphatase activity were observed. These results suggest that the intermittent shear of the order of 10 mPa can competently enhance osteogenic differentiation of rBMSCs compared to static culture.

Meniscal tissue engineering via 3D printed PLA monolith with carbohydrate based self-healing interpenetrating network hydrogel.

Failure of bioengineered meniscus implant after transplantation is a major concern owing to mechanical failure, lack of chondrogenic capability and patient specific design. In this article, we have, for the first time, fabricated a 3D printed scaffold with carbohydrate based self-healing interpenetrating network (IPN) hydrogels-based monolith construct for load bearing meniscus tissue. 3D printed PLA scaffold was surface functionalized and embedded with self-healing IPN hydrogel for interfacial bonding further characterized by micro CT. Using collagen (C), alginate (A) and oxidized alginate (ADA), we developed self-healing IPN hydrogels with dual crosslinking (Ca2+ based ionic crosslinking and Schiff base (A-A, A-ADA)) capability and studied their physicochemical properties. Further, we studied human stem cells behaviour and chondrogenic differentiation potential within these IPN hydrogels. In-vivo heterotopic implantation confirmed biocompatibility of the monolith showing the feasibility of using carbohydrate based IPN hydrogel embedded in 3D printed scaffold for meniscal tissue development.