In vitro and in vivo evaluation of cerium oxide nanoparticles in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis among children worldwide, yet there is no vaccine for RSV disease. This study investigates the potential of cube and sphere-shaped cerium oxide nanoparticles (CNP) to modulate reactive oxygen (ROS) and nitrogen (RNS) species and immune cell phenotypes in the presence of RSV infection in vitro and in vivo. Cube and sphere-shaped CNP were synthesized by hydrothermal and ultrasonication methods, respectively. Physico-chemical characterization confirmed the shape of sphere and cube CNP and effect of various parameters on their particle size distribution and zeta potential. In vitro results revealed that sphere and cube CNP differentially modulated ROS and RNS levels in J774 macrophages. Specifically, cube CNP significantly reduced RSV-induced ROS levels without affecting RNS levels while sphere CNP increased RSV-induced RNS levels with minimal effect on ROS levels. Cube CNP drove an M1 phenotype in RSV-infected macrophages in vitro by increasing macrophage surface expression of CD80 and CD86 with a concomitant increase in TNFα and IL-12p70, while simultaneously decreasing M2 CD206 expression. Intranasal administration of sphere and cube-CNP were well-tolerated with no observed toxicity in BALB/c mice. Notably, cube CNP preferentially accumulated in murine alveolar macrophages and induced their activation, avoiding enhanced uptake and activation of other inflammatory cells such as neutrophils, which are associated with RSV-mediated inflammation. In conclusion, we report that sphere and cube CNP modulate macrophage polarization and innate cellular responses during RSV infection.

The Incidence, Degree, and Timing of Hypocalcemia From Massive Transfusion: A Retrospective Review.

BACKGROUND: Electrolyte administration during massive transfusion without readily available calcium laboratory values is likely ubiquitous but not well standardized. We aimed to quantify the incidence, degree, and timing of hypocalcemia during the first 24 hours after initiation of a massive transfusion with the institutional massive transfusion protocol (MTP). We hypothesized that hypocalcemia is prevalent during acute resuscitation (first six hours) despite efforts of the treatment team to replete calcium during active resuscitation. METHODS: A retrospective chart review of all patients who underwent MTP at our institution between January 1, 2017, and December 31, 2017, was performed. The primary outcome was hypocalcemia from a massive transfusion during the first six hours after the initiation of the MTP. Secondary outcomes of interest included hypercalcemia, hypomagnesemia, hospital mortality, peak and nadir timing of hypocalcemia and hypercalcemia, calcium supplementation, and calcium supplementation timing. Calcium administration and blood product transfusion is reported relative to the start of the MTP. The association between the total amount of calcium administered and the total number of blood products transfused was assessed. RESULTS: Data from 52 massive transfusions were analyzed. Ninety-seven percent of patients were hypocalcemic during the first six hours of resuscitation. The nadir occurred after median of eight units of blood product were given, (interquartile range {IQR}: 4-16). Calcium supplementation correlated with the total number of blood products transfused (ρ = 0.47, p < 0.01). Patients in whom calcium was supplemented received more blood products when compared to patients in whom calcium was not supplemented (median: 16, IQR: 12-26 vs. median: 9, IQR: 7-12, p <0.01). CONCLUSIONS: Hypocalcemia from massive transfusion is common. The incidence of hypocalcemia in MTP has been reported to be 85-97%. Calcium supplementation that is not standardized in MTP may lead to underutilization during massive transfusion and to hypocalcemia in these patients.

Chemokine-Based Therapeutics for the Treatment of Inflammatory and Fibrotic Convergent Pathways in COVID-19.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the SARS-CoV-2 betacoronavirus and has taken over 761,426 American lives as of the date of publication and will likely result in long-term, if not permanent, tissue damage for countless patients. COVID-19 presents with diverse and multisystemic pathologic processes, including a hyperinflammatory response, acute respiratory distress syndrome (ARDS), vascular injury, microangiopathy, tissue fibrosis, angiogenesis, and widespread thrombosis across multiple organs, including the lungs, heart, kidney, liver, and brain. C-X-C chemokines contribute to these pathologies by attracting inflammatory mediators, the disruption of endothelial cell integrity and function, and the initiation and propagation of the cytokine storm. Among these, CXCL10 is recognized as a critical contributor to the hyperinflammatory state and poor prognosis in COVID-19. CXCL10 is also known to regulate growth factor-induced fibrosis, and recent evidence suggests the CXCL10-CXCR3 signaling system may be vital in targeting convergent pro-inflammatory and pro-fibrotic pathways. This review will explore the mechanistic role of CXCL10 and related chemokines in fibrotic complications associated with COVID-19 and the potential of CXCL10-targeted therapeutics for early intervention and long-term treatment of COVID-19-induced fibrosis.

Prognostic Significance of Nonischemic Myocardial Fibrosis in Patients With Normal LV Volumes and Ejection-Fraction.

OBJECTIVES: This study aims to investigate the prognostic significance of late gadolinium enhancement (LGE) in patients without coronary artery disease and with normal range left ventricular (LV) volumes and ejection fraction. BACKGROUND: Nonischemic patterns of LGE with normal LV volumes and ejection fraction are increasingly detected on cardiovascular magnetic resonance, but their prognostic significance, and consequently management, is uncertain. METHODS: Patients with midwall/subepicardial LGE and normal LV volumes, wall thickness, and ejection fraction on cardiovascular magnetic resonance were enrolled and compared to a control group without LGE. The primary outcome was actual or aborted sudden cardiac death (SCD). RESULTS: Of 748 patients enrolled, 401 had LGE and 347 did not. The median age was 50 years (interquartile range: 38-61 years), LV ejection fraction 66% (interquartile range: 62%-70%), and 287 (38%) were women. Scan indications included chest pain (40%), palpitation (33%) and breathlessness (13%). No patient experienced SCD and only 1 LGE+ patient (0.13%) had an aborted SCD in the 11th follow-up year. Over a median of 4.3 years, 30 patients (4.0%) died. All-cause mortality was similar for LGE+/- patients (3.7% vs 4.3%; P = 0.71) and was associated with age (HR: 2.04 per 10 years; 95% CI: 1.46-2.79; P < 0.001). Twenty-one LGE+ and 4 LGE- patients had an unplanned cardiovascular hospital admission (HR: 7.22; 95% CI: 4.26-21.17; P < 0.0001). CONCLUSIONS: There was a low SCD risk during long-term follow-up in patients with LGE but otherwise normal LV volumes and ejection fraction. Mortality was driven by age and not LGE presence, location, or extent, although the latter was associated with greater cardiovascular hospitalization for suspected myocarditis and symptomatic ventricular tachycardia.

Assessment of 18F-PBR-111 in the Cuprizone Mouse Model of Multiple Sclerosis.

The study aims to assess site assessment of the performance of 18F-PBR-111 as a neuroinflammation marker in the cuprizone mouse model of multiple sclerosis (MS). 18F-PBR-111 PET imaging has not been well evaluated in multiple sclerosis applications both in preclinical and clinical research. This study will help establish the potential utility of 18F-PBR-111 PET in preclinical MS research and future animal and future human applications. 18F-PBR-111 PET/CT was conducted at 3.5 weeks (n = 7) and 5.0 weeks (n = 7) after cuprizone treatment or sham control (n = 3) in the mouse model. A subgroup of mice underwent autoradiography with cryosectioned brain tissue. T2 weighted MRI was performed to obtain the brain structural data of each mouse. 18F-PBR-111 uptake was assessed in multiple brain regions with PET and autoradiography images. The correlation between autoradiography and immunofluorescence staining of neuroinflammation (F4/80 and CD11b) was measured. Compared to control mice, significant 18F-PBR-111 uptake in the corpus callosum (p < 0.001), striatum (caudate and internal capsule, p < 0.001), and hippocampus (p < 0.05) was identified with PET images at both 3.5 weeks and 5.0 weeks, and validated with autoradiography. No significant uptake differences were detected between 3.5 weeks and 5.0 weeks assessing these regions as a whole, although there was a trend of increased uptake at 5.0 weeks compared to 3.5 weeks in the CC. High 18F-PBR-111 uptake regions correlated with microglial/macrophage locations by immunofluorescence staining with F4/80 and CD11b antibodies. 18F-PBR-111 uptake in anatomic locations correlated with activated microglia at histology in the cuprizone mouse model of MS suggests that 18F-PBR-111 has potential for in vivo evaluation of therapy response and potential for use in MS patients and animal studies.

Autonomous Care Pathway to Patient Opioid Abstinence: Should All Programs Offer this Approach?

Introduction: The opioid epidemic resulted in vast increase in neonatal opioid withdrawal syndrome (NOWS). To mitigate NOWS and opioid dependency among women, staff established a gender specific, patient driven, autonomy based, outpatient therapeutic substitution program. Methods: Prospective observational study of obstetric patients receiving prenatal care 7/1/2016-12/31/2019. Patients underwent universal urine drug screens to identify illicit drug use with dependency and offered addiction counseling with voluntary outpatient therapeutic substitution in an obstetrical-addictions combined clinic to achieve abstinence with oral Buprenorphine tapering protocol. Urine substance screening and cord blood testing were obtained at delivery. Birth outcomes compared among groups who achieved abstinence at birth, were successful at tapering, or continued opioid use. Results: Of 783 births, 165 (20.9%) demonstrated opioid use with 91 (55.2%) participating at some point in pregnancy in therapeutic substitution program. At birth, 14/94 (14.9%) patients completed the program and achieved opioid abstinence, 22/94 (23.4%) still enrolled and actively tapering. 57/94 (34.5%) patients were lost to follow-up, relapsed, or terminated due to non-compliance. Seventy-four of 67 (44.3%) opioid positive mothers chose not to enroll. Of 14 women who completed the program, 0 babies born with NOWS, compared to 11/22 (50%) still enrolled in program and actively tapering, 29/57 (50.9%) lost to follow-up, relapsed, or terminated due to non-compliance, and 28/74 (37.8%) never enrolled in program. Conclusion/Implications: Outpatient therapeutic substitution with oral Buprenorphine with abstinence is possible in pregnant patients and results zero NOWS. More data are needed to confirm findings and explore methods for enhanced success in obtaining abstinence. Support: Appalachian Regional Commission and Prevention (ARC) 1st through Charleston Area Medical Center in cooperation with Charleston Health Education and Research Institute (CHERI).

Liposomal Bupivacaine Versus Bupivacaine for Intercostal Nerve Blocks in Thoracic Surgery: A Retrospective Analysis.

BACKGROUND: Liposomal bupivacaine (LipoB), delivered via intercostal nerve blocks (ICNBs), is increasingly being used for postoperative pain control in thoracic surgery patients, but there is limited data on its effectiveness when compared to standard bupivacaine. OBJECTIVE: We sought to compare postoperative opioid use, pain control, and length of stay (LOS) in patients undergoing thoracic surgery with LipoB ICNBs vs patients undergoing thoracic surgery with ICNBs using standard bupivacaine. STUDY DESIGN: A retrospective analysis. SETTING: Research took place in a tertiary academic medical center. METHODS: A transition in the standard of care from standard bupivacaine to LipoB for ICNBs in March of 2014 allowed us to compare 2 cohorts: patients who received bupivacaine ICNBs from January 2013 through February of 2014 and patients who received LipoB ICNBs from March 2015 through November 2017. We included patients who underwent thoracic surgery for lung cancer using robotic-assisted thoracic surgery (RATS), video-assisted thoracic surgery (VATS), or traditional open thoracotomy, and documentation of ICNB in the operative note. We collected data on pain scores (Visual Analog Scale [VAS]) and opioid consumption (converted to oral morphine equivalents [OMEs]) intraoperatively, on postoperative day (POD) 0, POD 1, POD 2, and POD 3. We also analyzed data on length of stay [LOS]. A primary analysis was performed on the effects of LipoB vs bupivacaine across all surgery types on opioid consumption, pain scores, and LOS with a secondary analysis on the same endpoints per individual surgery type. RESULTS: A total of 129 patients were included from the predefined study periods (n = 62 LipoB and n = 67 standard bupivacaine). Across all surgery types, LipoB decreased opioid utilization vs standard bupivacaine (P < .01). Post-hoc testing revealed that this difference existed intraoperatively (55 ± 5 vs 69 ± 4 mg OME, P = .03) and on POD 0 (44 ± 6 vs 68 ± 6 mg OME, P < .01). Surgical subtype analysis revealed that this difference was mostly driven by lower opioid consumption in patients undergoing RATS. When compared across all surgery types, LipoB vs bupivacaine did not affect postoperative pain scores. However, subgroup analysis showed that pain scores were lower in the LipoB vs standard bupivacaine group undergoing VATS on POD 0, 1, and 2. The LOS across all thoracic surgery types was lower in the LipoB group when compared to the standard bupivacaine group (median, 4 days [IQR 2.0-6.0] vs median, 5 days [IQR 3.0-8.0], P < .01). Subgroup analysis showed that the LOS in patients undergoing VATS with LipoB ICNBs was shorter compared to patients receiving bupivacaine ICNBs. LIMITATIONS: The retrospective nature of this study makes it prone to several types of bias. CONCLUSION: ICNBs with LipoB for thoracic surgery leads to lower opioid consumption and shorter LOS when compared to ICNBs with standard bupivacaine. The benefit of LipoB over standard bupivacaine for ICNBs appears especially relevant in VATS or RATS procedures. KEY WORDS: Intercostal nerve block, liposomal bupivacaine, RATS, regional anesthesia, robotic-assisted thoracoscopic surgery, thoracotomy, VATS, video-assisted thoracoscopic surgery.

Design and evaluation of collagen-inspired mineral-hydrogel nanocomposites for bone regeneration.

Bone loss due to trauma and tumors remains a serious clinical concern. Due to limited availability and disease transmission risk with autografts and allografts, calcium phosphate bone fillers and growth factor-based substitute bone grafts are currently used in the clinic. However, substitute grafts lack bone regeneration potential when used without growth factors. When used along with the added growth factors, they lead to unwanted side effects such as uncontrolled bone growth. Collagen-based hydrogel grafts available on the market fail to provide structural guidance to native cells due to high water-solubility and faster degradation. To overcome these limitations, we employed bioinspired material design and fabricated three different hydrogels with structural features similar to native collagen at multiple length-scales. These hydrogels fabricated using polyionic complexation of oppositely charged natural polysaccharides exhibited multi-scale architecture mimicking nanoscale banding pattern, and microscale fibrous structure of native collagen. All three hydrogels promoted biomimetic apatite-like mineral deposition in vitro elucidating crystalline structure on the surface while amorphous calcium phosphate inside the hydrogels resulting in mineral-hydrogel nanocomposites. When evaluated in a non-load bearing critical size mouse calvaria defect model, chitosan - kappa carrageenan mineral-hydrogel nanocomposites enhanced bone regeneration without added growth factors compared to empty defect as well as widely used marketed collagen scaffolds. Histological assessment of the regenerated bone revealed improved healing and tissue remodeling with mineral-hydrogel nanocomposites. Overall, these collagen-inspired mineral-hydrogel nanocomposites showed multi-scale hierarchical structure and can potentially serve as promising bioactive hydrogel to promote bone regeneration. STATEMENT OF SIGNIFICANCE: Hydrogels, especially collagen, are widely used in bone tissue engineering. Collagen fibrils play arguably the most important role during natural bone development. Its multi-scale hierarchical structure to form fibers from fibrils and electrostatic charges enable mineral sequestration, nucleation, and growth. However, bulk collagen hydrogels exhibit limited bone regeneration and are mostly used as carriers for highly potent growth factors such as bone morphogenic protein-2, which increase the risk of uncontrolled bone growth. Thus, there is an unmet clinical need for a collagen-inspired biomaterial that can recreate structural hierarchy, mineral sequestration ability, and stimulate recruitment of host progenitor cells to facilitate bone regeneration. Here, we propose collagen-inspired bioactive mineral-hydrogel nanocomposites as a growth factor-free approach to guide and enhance bone regeneration.

Fibrillar Collagen Quantification With Curvelet Transform Based Computational Methods.

Quantification of fibrillar collagen organization has given new insight into the possible role of collagen topology in many diseases and has also identified candidate image-based bio-markers in breast cancer and pancreatic cancer. We have been developing collagen quantification tools based on the curvelet transform (CT) algorithm and have demonstrated this to be a powerful multiscale image representation method due to its unique features in collagen image denoising and fiber edge enhancement. In this paper, we present our CT-based collagen quantification software platform with a focus on new features and also giving a detailed description of curvelet-based fiber representation. These new features include C++-based code optimization for fast individual fiber tracking, Java-based synthetic fiber generator module for method validation, automatic tumor boundary generation for fiber relative quantification, parallel computing for large-scale batch mode processing, region-of-interest analysis for user-specified quantification, and pre- and post-processing modules for individual fiber visualization. We present a validation of the tracking of individual fibers and fiber orientations by using synthesized fibers generated by the synthetic fiber generator. In addition, we provide a comparison of the fiber orientation calculation on pancreatic tissue images between our tool and three other quantitative approaches. Lastly, we demonstrate the use of our software tool for the automatic tumor boundary creation and the relative alignment quantification of collagen fibers in human breast cancer pathology images, as well as the alignment quantification of in vivo mouse xenograft breast cancer images.

Self-assembly of multiscale anisotropic hydrogels through interfacial polyionic complexation.

Polysaccharides are explored for various tissue engineering applications due to their inherent cytocompatibility and ability to form bulk hydrogels. However, bulk hydrogels offer poor control over their microarchitecture and multiscale hierarchy, parameters important to recreate extracellular matrix-mimetic microenvironment. Here, we developed a versatile platform technology to self-assemble oppositely charged polysaccharides into multiscale fibrous hydrogels with controlled anisotropic microarchitecture. We employed polyionic complexation through microfluidic flow of positively charged polysaccharide, chitosan, along with one of the three negatively charged polysaccharides: alginate, gellan gum, and kappa carrageenan. These hydrogels were composed of microscale fibers, which in turn were made of submicron fibrils confirming multiscale hierarchy. Fibrous hydrogels showed strong tensile mechanical properties, which were further modulated by encapsulation of shape-specific antioxidant cerium oxide nanoparticles (CNPs). Specifically, hydrogels with chitosan and gellan gum showed more than eight times higher tensile strength compared to the other two pairs. Incorporation of sphere-shaped cerium oxide nanoparticles in chitosan and gellan gum further reinforced fibrous hydrogels and increased their tensile strength by 40%. Altogether, our automated hydrogel fabrication platform allows fabrication of bioinspired biomaterials with scope for one-step encapsulation of small molecules and nanoparticles without chemical modification or use of chemical crosslinkers.

Bottom-Up Self-assembled Hydrogel-Mineral Composites Regenerate Rabbit Ulna Defect without Added Growth Factors.

Hydrogel-based biomaterials have advanced bone tissue engineering approaches in the last decade, through their ability to serve as a carrier for potent growth factor, bone morphogenic protein-2 (BMP-2). However, biophysical properties of hydrogels such as multiscale structural hierarchy and bone extracellular matrix (ECM)-mimetic microarchitecture are underutilized while designing current bone grafts. Incorporation of these properties offers great potential to create a favorable biomimetic microenvironment to harness their regenerative potential. Here, we present our approach to fabricate collagen-inspired bioactive hydrogel scaffolds (referred to as "RegenMatrix") to guide and enhance bone regeneration in a rabbit ulna defect model through the mimicry of multiscale architecture of bone ECM, i.e., native collagen. Specifically, we employed polyelectrolyte complexation to promote bottom-up self-assembly of oppositely charged polysaccharides (chitosan and kappa-carrageenan) at multiple length scales forming fibrils, which further assemble into fibers. The self-assembly and bioinspired scaffold fabrication method resulted in robust cylindrical RegenMatrix with excellent retention of the multiscale architecture and uniform mineral deposition throughout the scaffolds. RegenMatrix, in both nonmineralized and mineralized forms, enhanced bone regeneration in the semiload-bearing ulna defect when compared to the empty defect. RegenMatrix also showed greater histocompatibility without any fibrous tissue formation. Collectively, the RegenMatrix developed in this study has a great potential as a bioactive bone graft without any added growth factors.

Hierarchically aligned fibrous hydrogel films through microfluidic self-assembly of graphene and polysaccharides.

Despite significant interest in developing extracellular matrix (ECM)-inspired biomaterials to recreate native cell-instructive microenvironments, the major challenge in the biomaterial field is to recapitulate the complex structural and biophysical features of native ECM. These biophysical features include multiscale hierarchy, electrical conductivity, optimum wettability, and mechanical properties. These features are critical to the design of cell-instructive biomaterials for bioengineering applications such as skeletal muscle tissue engineering. In this study, we used a custom-designed film fabrication assembly, which consists of a microfluidic chamber to allow electrostatic charge-based self-assembly of oppositely charged polymer solutions forming a hydrogel fiber and eventually, a nanocomposite fibrous hydrogel film. The film recapitulates unidirectional hierarchical fibrous structure along with the conductive properties to guide initial alignment and myotube formation from cultured myoblasts. We combined high conductivity, and charge carrier mobility of graphene with biocompatibility of polysaccharides to develop graphene-polysaccharide nanocomposite fibrous hydrogel films. The incorporation of graphene in fibrous hydrogel films enhanced their wettability, electrical conductivity, tensile strength, and toughness without significantly altering their elastic properties (Young's modulus). In a proof-of-concept study, the mouse myoblast cells (C2C12) seeded on these nanocomposite fibrous hydrogel films showed improved spreading and enhanced myogenesis as evident by the formation of multinucleated myotubes, an early indicator of myogenesis. Overall, graphene-polysaccharide nanocomposite fibrous hydrogel films provide a potential biomaterial to promote skeletal muscle tissue regeneration.

Effect of reduced glutathione supplementation on cryopreservation induced sperm cryoinjuries in Murrah bull semen.

The experiment was conducted to study cryopreservation induced sperm cryoinjuries and the protective effect of reduced Glutathione supplementation in Murrah bull semen. A total of 20 semen ejaculates were split into two parts after initial examination and were extended in glycerolated egg yolk TRIS diluter (Control group) and glycerolated egg yolk TRIS diluter + 0.5 mM reduced Glutathione (Treatment Group). The diluted semen samples were loaded into 0.25 ml French mini straw and sealing of straws were done. Thereafter, semen straws were kept for equilibration for 4 h at 5 °C and semen was frozen using a standard cryopreservation protocol in automatic biological freezer. Post-thaw analysis was performed after 24 h of semen storage in liquid nitrogen. Fresh and post-thaw sperm assessments included sperm motility, viability (SYBR-14/PI assay), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (HOST). Cryopreservation of semen in liquid nitrogen induced a marked reduction in post-thaw sperm motility, viability, mitochondrial function and plasma membrane integrity and increased moribund type of sperm (SYBR-14/PI assay) in control group as compared to reduced glutathione treated group. There were significant (P < 0.05) cryo injuries in frozen-thawed spermatozoa following cryopreservation in buffalo bull semen. The supplementation of reduced glutathione in treatment group exhibited significantly (P < 0.05) lower cryoinjuries during cryopreservation and semen storage in liquid nitrogen. From the study it was concluded that, spermatozoa from Murrah bulls are susceptible to injuries due to cryopreservation in liquid nitrogen, but these cryo induced damage can be protected significantly (P < 0.05) by the use of reduced Glutathione.

Synthesis, physico-chemical characterization, and antioxidant effect of PEGylated cerium oxide nanoparticles.

Cerium oxide nanoparticles (CNPs) represent a promising class of antioxidant nanoparticles with potential therapeutic value. Due to the easily reversible oxidation states of cerium (Ce3+ and Ce4+) at the nanoscale, CNPs scavenge excessive reactive oxygen and nitrogen species in a self-regenerative manner. In this study, we have demonstrated a simple method to functionalize shape-specific CNPs (i.e., rod- and cube-shaped) with polyethylene glycol (PEG) and studied the effect of PEGylation on the physico-chemical properties, antioxidant activity, and biocompatibility of rod- and cube-shaped CNPs. The chemical conjugation of PEG onto the CNP surface was confirmed by a series of physico-chemical characterizations (1H-NMR, FTIR, and surface zeta potential). Rod-shaped CNPs demonstrated greater reactive oxygen species scavenging ability compared to cube-shaped CNPs. PEGylation of CNPs did not affect shape, cerium oxidation state, and cytocompatibility. Importantly, PEGylation significantly reduced the amount of proteins adsorbed onto the CNPs. The antioxidant effects of CNPs were maintained in PEGylated CNPs. We envision that PEGylated rod-shaped CNPs synthesized in this study have the potential to be biocompatible nanoparticles that can combat oxidative stress-related diseases.

Hypoxic tumor microenvironment: Opportunities to develop targeted therapies.

In recent years, there has been great progress in the understanding of tumor biology and its surrounding microenvironment. Solid tumors create regions with low oxygen levels, generally termed as hypoxic regions. These hypoxic areas offer a tremendous opportunity to develop targeted therapies. Hypoxia is not a random by-product of the cellular milieu due to uncontrolled tumor growth; rather it is a constantly evolving participant in overall tumor growth and fate. This article reviews current trends and recent advances in drug therapies and delivery systems targeting hypoxia in the tumor microenvironment. In the first part, we give an account of important physicochemical changes and signaling pathways activated in the hypoxic microenvironment. This is then followed by various treatment strategies including hypoxia-sensitive signaling pathways and approaches to develop hypoxia-targeted drug delivery systems.

Cell-Instructive Graphene-Containing Nanocomposites Induce Multinucleated Myotube Formation.

Myoblast differentiation is a key step in myogenesis and has long been considered to be controlled mainly by biochemical cues such as growth factors. However, the tissue engineering approaches based on biochemical cues demonstrate low reproducibility as a precise spatial control over their bioactivity is challenging. Recently, substrate micro/nano-structure and electro-responsive properties are recognized for their important roles in myoblast differentiation. In this study, we hypothesized that engineering biophysical features such as nano/micro-fibrous structure and conductive properties into a single biomaterial scaffold will instruct the myoblasts to differentiate into multinucleated myotubes even in the absence of differentiation media. We fabricated nanocomposite scaffolds composed of conductive graphene nanosheets and polycaprolactone (PCL), a widely used biocompatible material. The resulting graphene-PCL scaffolds possess excellent conductivity due to graphene nanosheets and great processability, biodegradability and elastic mechanical properties conferred by PCL. Additionally, physicochemical and mechanical properties of nanocomposite scaffolds can be tuned by varying graphene concentration. Further, graphene-PCL nanocomposites and their 8-week degradation products exhibited remarkable cytocompatibility and promoted adhesion and proliferation of C2C12 mouse myoblast cells. Importantly, these nanocomposite scaffolds induced graphene concentration-dependent differentiation of C2C12 cells into multinucleated myotubes even in normal growth media suggesting their cell-instructive potential. Thus, graphene-PCL nanocomposite scaffolds can serve as a strategy to promote skeletal muscle regeneration without biochemical cues.

Carbon-based hierarchical scaffolds for myoblast differentiation: Synergy between nano-functionalization and alignment.

While several scaffolds have been proposed for skeletal muscle regeneration, multiscale hierarchical scaffolds with the complexity of extracellular matrix (ECM) haven't been engineered successfully. By precise control over nano- and microscale features, comprehensive understanding of the effect of multiple factors on skeletal muscle regeneration can be derived. In this study, we engineered carbon-based scaffolds with hierarchical nano- and microscale architecture with controlled physico-chemical properties. More specifically, we built multiscale hierarchy by growing carbon nanotube (CNT) carpets on two types of scaffolds, namely, interconnected microporous carbon foams and aligned carbon fiber mats. Nanostructured CNT carpets offered fine control over nano-roughness and wettability facilitating myoblast adhesion, growth and differentiation into myocytes. However, microporous foam architecture failed to promote their fusion into multinucleated myotubes. On the other hand, aligned fibrous architecture stimulated formation of multinucleated myotubes. Most importantly, nanostructured CNT carpets interfaced with microscale aligned fibrous architecture significantly enhanced myocyte fusion into multinucleated mature myotubes highlighting synergy between nanoscale surface features and micro-/macroscale aligned fibrous architecture in the process of myogenesis. STATEMENT OF SIGNIFICANCE: Due to limited regenerative potential of skeletal muscle, strategies stimulating regeneration of functional muscles are important. These strategies are aimed at promoting differentiation of progenitor cells (myoblasts) into multinucleated myotubes, a key initial step in functional muscle regeneration. Recent tissue engineering approaches utilize various scaffolds ranging from decellularized matrices to aligned biomaterial scaffolds. Although, majority of them have focused on nano- or microscale organization, a systematic approach to build the multiscale hierarchy into these scaffolds is lacking. Here, we engineered multiscale hierarchy into carbon-based materials and demonstrated that the nanoscale features govern the differentiation of individual myoblasts into myocytes whereas microscale alignment cues orchestrate fusion of multiple myocytes into multinucleated myotubes underlining the importance of multiscale hierarchy in enhancing coordinated tissue regeneration.

Effect of supplementation of butylated hydroxytoluene on post-thaw sperm viability, motility and membrane integrity of Hariana bulls.

AIM: This study was aimed to see the beneficial effect of butylated hydroxytoluene (BHT) as a semen additive of Hariana bull semen. MATERIALS AND METHODS: The study was carried out in Hariana bulls. Twenty-four ejaculates from two bulls were used for this study. Each ejaculate was extended with standard glycerolated egg yolk tris extender and supplemented with BHT at two concentrations as 0.5 mM (T1) and 1.0 mM (T2). After dilution, equilibration and 24 h of cryopreservation, the samples were analyzed for progressive motility, sperm viability and membrane integrity. RESULTS: Progressive motility, sperm viability and sperm membrane integrity were significantly (p<0.05) increased in the samples fortified with BHT as compared to the control during the process of cryopreservation and thawing. The BHT concentration of 1 mM revealed better results as compared to 0.5 mM. CONCLUSION: Addition of 1.0 mM BHT was found better in cryopreservation of Hariana bull semen compared to 0.5 mM BHT and control samples. The addition of BHT has improved the sperm quality by acting as an antioxidant thereby reducing the lipid peroxidation of the sperms.