Nude Rats Research Articles

Application of Human Epineural Conduit Supported with Human Mesenchymal Stem Cells as a Novel Therapy for Enhancement of Nerve Gap Regeneration

Various therapeutic methods have been suggested to enhance nerve regeneration. In this study, we propose a novel approach for enhancement of nerve gap regeneration by applying human epineural conduit (hEC) supported with human mesenchymal stem cells (hMSC), as an alternative to autograft repair. Restoration of 20 mm sciatic nerve defect with hEC created from human sciatic nerve supported with hMSC was tested in 4 experimental groups (n = 6 each) in the athymic nude rat model (Crl:NIH-Foxn1rnu): 1 - No repair control, 2 - Autograft control, 3 - Matched diameter hEC filled with 1 mL saline, 4 - Matched diameter hEC supported with 3 × 106 hMSC. Assessments included: functional tests: toe-spread and pinprick, regeneration assessment by immunofluorescence staining: HLA-1, HLA-DR, NGF, GFAP, Laminin B, S-100, VEGF, vWF and PKH26 labeling; histomorphometric analysis of myelin thickness, axonal density, fiber diameter and myelinated nerve fibers percentage; Gastrocnemius Muscle Index (GMI) and muscle fiber area ratio. Best sensory and motor function recovery, as well as GMI and muscle fiber area ratio, were observed in the autograft group, and were comparable to the hEC with hMSC group (p = 0.038). Significant improvements of myelin thickness (p = 0.003), fiber diameter (p = 0.0296), and percentage of myelinated fibers (p < 0.0001) were detected in hEC group supported with hMSC compared to hEC with saline controls. At 12-weeks after nerve gap repair, hEC combined with hMSC revealed increased expression of neurotrophic and proangiogenic factors, which corresponded with improvement of function comparable with the autograft control. Application of our novel hEC supported with hMSC provides a potential alternative to the autograft nerve repair.Graphical

Abstract 12020: Heterogeneity and Plasticity of Cardiac Fibroblasts Governing Reparative Lymphangiogenesis After Myocardial Infarction: A New Therapeutic Approach for Heart Failure

Introduction: Cardiac lymphangiogenesis has attracted attention as a therapeutic target after myocardial infarction (MI). Lymphangiogenic remodeling has been observed after MI in adult and fetal hearts; however, reparative lymphangiogenic remodeling is only observed in fetal hearts. The factors that determine the fate of this phenomenon have not been fully elucidated. Hypothesis: We demonstrated that a specific population of VCAM1 + human fetal cardiac fibroblasts (fCFs) restore cardiac function post-MI by lymphangiogenesis. Thus, we hypothesize that adult cardiac fibroblasts (aCFs), compared to fCFs, possess a different distribution of fibroblasts with differing lymphangiogenic potential. Furthermore, we also hypothesize that aCFs can be exogenously manipulated to acquire fCFs-like reparative lymphangiogenic potential, which can be used as a cell therapy for heart failure. Methods: Flow cytometry assessed CD90 and VCAM1 expression of aCFs and fCFs. To shift aCFs towards a fCF phenotype, TNF-α and IL-4 were added to culture medium. aCF subpopulations were intramyocardially injected in nude rats and swine post-MI with subsequent echocardiography. Myocardial tissue staining (Sirius Red, LYVE1) and RNA-seq were performed to identify the molecular mechanism. Results: aCFs and fCFs exhibited different distributions of CD90 and VCAM1 expression, where aCFs showed lower CD90 and VCAM1 expression. The addition of TNF-α and IL-4 shifted the localization of VCAM1 + aCFs towards a fCF distribution by activation of NF-κB. VCAM1 + CD90 + fCFs-like aCFs provided a sustained improvement in left ventricular ejection fraction and showed reduced fibrosis and increased lymphangiogenesis. This effect was recapitulated in a large animal model. In terms of the molecular mechanism, 13 candidate genes were identified. Conclusions: These findings suggest that the heterogenous and plastic polarity of aCFs and fCFs determines the fate of the lymphangiogenic response after MI and that this response can be regulated by 13 genes. This artificial creation of the VCAM1 + fCFs-like fibroblast environment after MI has enabled a clinical trial for a new cell therapy for inducing reparative lymphangiogenesis (clinical trial ID: jRCT2033210078).

Abstract 11090: Therapeutic Effect of VEGF- and SDF-Nanoparticles in the Rat Monocrotaline Model of Pulmonary Hypertension

Introduction: Pulmonary vascular endothelial cell injury, dysfunction, and apoptosis are associated with the development of pulmonary arterial hypertension (PAH). Improving endothelial function may be therapeutically beneficial in this disorder. In the present study, we examined the effect of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α), proteins that support endothelial repair and tissue regeneration, in rats with monocrotaline-induced PAH. A polysaccharide nanoparticle was used as a carrier for VEGF and SDF-1α delivery, the formulation of which extends the retention time of the proteins in the lung by ~17-fold compared with free protein. Method: A crosslinked dextran sulfate-chitosan nanoparticle (XDSCS NP) was prepared as the carrier. Recombinant human VEGF and SDF-1α were incorporated in the XDSCS NP to generate VEGF-NP and SDF-NP, respectively. Athymic nude rats were injected with monocrotaline (50 mg/kg, IP) to induce PAH. Two weeks after the injection, VEGF-NP (8 μg/rat) and SDF-NP (4 μg/rat) were delivered to the lungs of rats via intratracheal aerosolization. At 4 weeks, catheterization of right and left heart was performed, and lung tissue collected for histological analysis. Results: Monocrotaline induced PAH in the nude rats, leading to right ventricular systolic pressure (RVSP) of 71±8 mmHg (mean±SD, n=8) vs. 29±2 mmHg in the untreated control rats. VEGF-NP and SDF-NP treatment reduced the RVSP to 48±13 mmHg. The Fulton indices of the three groups were 0.45±0.07, 0.22±0.01, and 0.25±0.06, respectively. The pulmonary vascular resistance indices of the groups were 3.2±0.7, 0.6±0.3, and 2.1±1.0 mmHg.min/ml/kg, respectively. Histological analysis showed monocrotaline-treated rats had prominent muscularization of precapillary vessels, which was markedly reduced by the VEGF-NP and SDF-NP treatment. Aerosolization of free VEGF and SDF-1α or of empty XDSCS NP carrier had no effect on monocrotaline-induce PAH. Conclusion: These data indicate that the delivery of VEGF and SDF to the lungs of rats in a NP-bound form had beneficial therapeutic effects in monocrotaline-induced PAH, and offer the potential for a novel therapeutic strategy for human pulmonary hypertensive disorders.

Effects of amyloid precursor protein peptide APP96-110, alone or with human mesenchymal stromal cells, on recovery after spinal cord injury.

Delivery of a peptide (APP96-110), derived from amyloid precursor protein (APP), has been shown to elicit neuroprotective effects following cerebral stroke and traumatic brain injury. In this study, the effect of APP96-110 or a mutant version of this peptide (mAPP96-110) was assessed following moderate (200 kdyn, (2 N)) thoracic contusive spinal cord injury (SCI) in adult Nude rats. Animals received a single tail vein injection of APP96-110 or mAPP96-110 at 30 minutes post-SCI and were then assessed for functional improvements over the next 8 weeks. A cohort of animals also received transplants of either viable or non-viable human mesenchymal stromal cells (hMSCs) into the SC lesion site at one week post-injury to assess the effect of combining intravenous APP96-110 delivery with hMSC treatment. Rats were perfused 8 weeks post-SCI and longitudinal sections of spinal cord analyzed for a number of factors including hMSC viability, cyst size, axonal regrowth, glial reactivity and macrophage activation. Analysis of sensorimotor function revealed occasional significant differences between groups using Ladderwalk or Ratwalk tests, however there were no consistent improvements in functional outcome after any of the treatments. mAPP96-110 alone, and APP96-110 in combination with both viable and non-viable hMSCs significantly reduced cyst size compared to SCI alone. Combined treatments with donor hMSCs also significantly increased βIII tubulin+, glial fibrillary acidic protein (GFAP+) and laminin+ expression, and decreased ED1+ expression in tissues. This preliminary study demonstrates that intravenous delivery of APP96-110 peptide has selective, modest neuroprotective effects following SCI, which may be enhanced when combined with hMSC transplantation. However, the effects are less pronounced and less consistent compared to the protective morphological and cognitive impact that this same peptide has on neuronal survival and behaviour after stroke and traumatic brain injury. Thus while the efficacy of a particular therapeutic approach in one CNS injury model may provide justification for its use in other neurotrauma models, similar outcomes may not necessarily occur and more targeted approaches suited to location and severity are required. All animal experiments were approved by The University of Western Australia Animal Ethics Committee (RA3/100/1460) on April 12, 2016.

Human iPS cell derived RPE strips for secure delivery of graft cells at a target place with minimal surgical invasion

Several clinical studies have been conducted into the practicality and safety of regenerative therapy using hESC/iPSC-retinal pigment epithelium (RPE) as a treatment for the diseases including age-related macular degeneration. These studies used either suspensions of RPE cells or an RPE cell sheet. The cells can be injected using a minimally invasive procedure but the delivery of an intended number of cells at an exact target location is difficult; cell sheets take a longer time to prepare, and the surgical procedure is invasive but can be placed at the target area. In the research reported here, we combined the advantages of the two approaches by producing a quickly formed hiPSC-RPE strip in as short as 2 days. The strip readily expanded into a monolayer sheet on the plate, and after transplantation in nude rats, it showed a potency to partly expand with the correct apical/basal polarity in vivo, although limited in expansion area in the presence of healthy host RPE. The strip could be injected into a target area in animal eyes using a 24G canula tip.

Anti-Tumor Immune Response and Long-Term Immunological Memory Induced by Minibeam Radiation Therapy: A Pilot Study

<h3>Purpose/Objective(s)</h3> Minibeam radiation therapy (MBRT) is a novel therapeutic approach that uses narrow beams (0.5 to 1 mm) to create a distinct spatial modulation of the dose, featuring alternating regions of high dose (peaks) and low dose (valleys). MBRT with both x-rays and protons was shown to provide a remarkable normal (skin and brain) tissue preservation in preclinical experiments, including sparing of cognitive, emotional, and motor processes. Equivalent or superior (glioma) tumor control compared to conventional radiation therapy was observed, and that even with highly heterogeneous dose distributions in small animal experiments. The underlying biological mechanisms under MBRT efficiency remain elusive. The aim of this work was to investigate the possible participation of the immune system in the anti-tumor response of MBRT. We also hypothesized the possible establishment of a long-term anti-tumor immunity. <h3>Materials/Methods</h3> We collected original <b>in vivo</b> radiopathological data in rats, thanks to the implementation of MBRT on a small animal radiation system. Firstly, we compared the response to MBRT of RG2-bearing nude (athymic) rats versus immunocompetent (Fischer) rats. The dose prescription was 30 Gy (average) in one fraction, with approximately one half of the tumor volume receiving (valley) doses of 6 Gy. Secondly, RG2-bearing rats having survived to the first part of the study were reinjected with the same tumor cell line and tumor development was monitored by MRI and bioluminescence imaging. <h3>Results</h3> While a significant increase of lifespan was observed in the irradiated immunocompetent rats with respect to non-irradiated controls, the irradiated nude rats showed no response to the treatment, pointing out a determinant role of T-cells in MBRT efficiency. Quantification of immune cells is ongoing. Additionally, the irradiated and cured immunocompetent RG2-bearing rats were rechallenged with RG2 tumor cells (3 to 6 months after irradiation). While naïve controls developed tumor normally, this was not the case for any of the previously irradiated animals. <h3>Conclusion</h3> These results suggest that T cells play a central role in tumor cell killing in MBRT. Moreover, MBRT seems to act as an anti-tumor vaccine. The remarkable reduction in neurotoxicity along with the potential activation of the immune system makes MBRT an ideal candidate to achieve an effective radio-immunotherapy in very infiltrative and immunosuppressive brain tumors, such as high-grade gliomas (GBM).

Matured Myofibers in Bioprinted Constructs with In Vivo Vascularization and Innervation.

For decades, the study of tissue-engineered skeletal muscle has been driven by a clinical need to treat neuromuscular diseases and volumetric muscle loss. The in vitro fabrication of muscle offers the opportunity to test drug-and cell-based therapies, to study disease processes, and to perhaps, one day, serve as a muscle graft for reconstructive surgery. This study developed a biofabrication technique to engineer muscle for research and clinical applications. A bioprinting protocol was established to deliver primary mouse myoblasts in a gelatin methacryloyl (GelMA) bioink, which was implanted in an in vivo chamber in a nude rat model. For the first time, this work demonstrated the phenomenon of myoblast migration through the bioprinted GelMA scaffold with cells spontaneously forming fibers on the surface of the material. This enabled advanced maturation and facilitated the connection between incoming vessels and nerve axons in vivo without the hindrance of a scaffold material. Immunohistochemistry revealed the hallmarks of tissue maturity with sarcomeric striations and peripherally placed nuclei in the organized bundles of muscle fibers. Such engineered muscle autografts could, with further structural development, eventually be used for surgical reconstructive purposes while the methodology presented here specifically has wide applications for in vitro and in vivo neuromuscular function and disease modelling.

Safety and efficacy of human juvenile chondrocyte-derived cell sheets for osteochondral defect treatment

Knee cartilage does not regenerate spontaneously after injury, and a gold standard regenerative treatment algorithm has not been established. This study demonstrates preclinical safety and efficacy of scaffold-free, human juvenile cartilage-derived-chondrocyte (JCC) sheets produced from routine surgical discards using thermo-responsive cultureware. JCCs exhibit stable and high growth potential in vitro over passage 10, supporting possibilities for scale-up to mass production for commercialization. JCC sheets contain highly viable, densely packed cells, show no anchorage-independent cell growth, express mesenchymal surface markers, and lack MHC II expression. In nude rat focal osteochondral defect models, stable neocartilage formation was observed at 4 weeks by JCC sheet transplantation without abnormal tissue growth over 24 weeks in contrast to the nontreatment group showing no spontaneous cartilage repair. Regenerated cartilage was safranin-O positive, contained type II collagen, aggrecan, and human vimentin, and lacked type I collagen, indicating that the hyaline-like neocartilage formed originates from transplanted JCC sheets rather than host-derived cells. This study demonstrates the safety of JCC sheets and stable hyaline cartilage formation with engineered JCC sheets utilizing a sustainable tissue supply. Cost-benefit and scaling issues for sheet fabrication and use support feasibility of this JCC sheet strategy in clinical cartilage repair.

Proof of concept of a multimodal intravital molecular imaging system for tumour transpathology investigation

BackgroundTranspathology highlights the interpretation of the underlying physiology behind molecular imaging. However, it remains challenging due to the discrepancies between in vivo and in vitro measurements and difficulties of precise co-registration between trans-scaled images. This study aims to develop a multimodal intravital molecular imaging (MIMI) system as a tool for in vivo tumour transpathology investigation.MethodsThe proposed MIMI system integrates high-resolution positron imaging, magnetic resonance imaging (MRI) and microscopic imaging on a dorsal skin window chamber on an athymic nude rat. The window chamber frame was designed to be compatible with multimodal imaging and its fiducial markers were customized for precise physical alignment among modalities. The co-registration accuracy was evaluated based on phantoms with thin catheters. For proof of concept, tumour models of the human colorectal adenocarcinoma cell line HT-29 were imaged. The tissue within the window chamber was sectioned, fixed and haematoxylin–eosin (HE) stained for comparison with multimodal in vivo imaging.ResultsThe final MIMI system had a maximum field of view (FOV) of 18 mm × 18 mm. Using the fiducial markers and the tubing phantom, the co-registration errors are 0.18 ± 0.27 mm between MRI and positron imaging, 0.19 ± 0.22 mm between positron imaging and microscopic imaging and 0.15 ± 0.27 mm between MRI and microscopic imaging. A pilot test demonstrated that the MIMI system provides an integrative visualization of the tumour anatomy, vasculatures and metabolism of the in vivo tumour microenvironment, which was consistent with ex vivo pathology.ConclusionsThe established multimodal intravital imaging system provided a co-registered in vivo platform for trans-scale and transparent investigation of the underlying pathology behind imaging, which has the potential to enhance the translation of molecular imaging.

A Prevascularized Polyurethane-Reinforced Fibrin Patch Improves Regenerative Remodeling in a Rat Right Ventricle Replacement Model.

Congenital heart defects (CHDs) affect 1 in 120 newborns in the United States. Surgical repair of structural heart defects often leads to arrhythmia and increased risk of heart failure. The laboratory has previously developed an acellular fibrin patch reinforced with a biodegradable poly(ether ester urethane) urea mesh that result in improved heart function when tested in a rat right ventricle wall replacement model compared to fixed pericardium. However, this patch does not drive significant neotissue formation. The patch materials are modified here and this patch is prevascularized with human umbilical vein endothelial cells and c-Kit+ human amniotic fluid stem cells. Rudimentary capillary-like networks form in the fibrin after culture of cell-encapsulated patches for 3 d in vitro. Prevascularized patches and noncell loaded patch controls are implanted onto full-thickness heart wall defects created in the right ventricle of athymic nude rats. Two months after surgery, defect repair with prevascularized patches results in improved heart function and the patched heart area exhibited greater vascularization and muscularization, less fibrosis, and increased M2 macrophage infiltration compared to acellular patches.

Acoustic vaporization of internalized superheated perfluorocarbon nanodroplets for imaging and macrophage function modulation

Ultrasound (US) can detect and interact with its contrast agents, allowing perfluorocarbon-based nanodroplets (NDs) or microbubbles (MBs) to detect and treat tumors in deep tissues. Since NDs given IV passively accumulate in tumor macrophages, we aimed to determine whether phagocytosed perfluorobutane (PFB) NDs that boil at -2 °C, can be vaporized in vivo using a clinical scanner. Should that be possible, PFB NDs can then be used to deliver drugs or genes to modulate macrophage function. We formulated stable PFB NDs and validated their internalization into THP-1 macrophages using confocal microscopy. PFB ND loaded macrophages were then infused into nude rats and US imaging (low MI US imaging, 18H6 transducer) of the liver and spleen performed in vivo using a Siemens Sequoia clinical scanner before and after applying MB destruction pulses (at 1.4 MI, 10L4 transducer). PFB ND-loaded macrophages were not visible using low MI imaging, before applying MB destruction pulses. They immediately vaporized into US visible microbubbles when destruction pulses were applied over the liver and remained visible for many minutes after vaporization. Our current focus is to assess ND-loaded macrophage function before and after ND activation to determine the feasibility of modulating immune function. [Study supported by CPRIT RR150010. RFM is a CPRIT Established Investigator. Siemens ACUSON Sequoia Ultrasound System provided as a loan by Siemens Medical Solutions, USA.]

Abstract 26: IL-17 Causes Hypertension, Reduced Fetal Weight And Natural Killer Cell Activation In The Absence Of T Cells

Preeclampsia (PE) is characterized by new onset hypertension (HTN), intrauterine growth restriction (IUGR), multi-organ dysfunction, and is associated with increased inflammatory cytokines, such as interleukin 17 (IL-17). More recent studies demonstrate a role for mitochondrial (mt) dysfunction/mtROS in the pathogenesis of PE. Although we have shown T helper cells from a rat model of PE cause HTN and mt dysfunction the causative factors for mt dysfunction are still being identified. In addition, we have shown that IL-17 cause HTN, IUGR and activate natural killer (NK) cells, and cause mt dysfunction in pregnant Sprague Dawley rats. However, in our previous studies we couldn’t differentiate the effect of activated TH cells versus IL-17 to cause these characteristics of PE. The athymic nude rat model lacks mature T cells but does have other components of the immune system, and will thus allow us to examine the role of IL-17 in the absence of TH cells in the pathophysiology of PE. We hypothesize that in the absence of T cells IL-17 induces HTN, NK cell activation and IUGR which is associated with renal and placental mt dysfunction during pregnancy. To test our hypothesis, IL-17 (150 pg/day) was infused via osmotic minipumps inserted on gestation day (GD) 14. Blood pressure (MAP) and mt function were measured on GD19 and were compared to untreated pregnant (NP) athymic nude rats. In response to IL-17; MAP increased from 95±4mmHg in NP(n=6) to 115±2 in NP+IL-17(n=6) (p&lt;0.001); pup weight decreased from 1.46±0.2 g in NP (n=6) to 0.98±0.07g in NP+IL-17 (n=6) (p&lt;0.05); NK cell activation increased from 0±0 %lymphocytes in NP (n=3) to 0.4±0.1% lymphocytes in NP+IL-17 rats (n=6). Interestingly, placental mtROS reduced 54% fold compared to NP and renal mtROS reduced 51.2% compared to NP. ATP production increased from 15.53±1.6 pmol of O2/sec/mg in NP (n=3) to 105.5±91 pmol of O2/sec/mg in NP+IL-17 (n=3) in the placenta, and from 1196±460 pmol of O2/sec/mg in NP (n=4) to 2016±951 pmol of O2/sec/mg NP+IL17 (n=4) in the kidney. These results show that although IL-17 induces HTN, IUGR, and NK cell activation independent of T cells, T cells are necessary for reduced mitochondrial function observed in PE and in rat models of placental ischemia.

MiR-4732-3p in Extracellular Vesicles From Mesenchymal Stromal Cells Is Cardioprotective During Myocardial Ischemia.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are an emerging alternative to cell-based therapies to treat many diseases. However, the complexity of producing homogeneous populations of EVs in sufficient amount hampers their clinical use. To address these limitations, we immortalized dental pulp-derived MSC using a human telomerase lentiviral vector and investigated the cardioprotective potential of a hypoxia-regulated EV-derived cargo microRNA, miR-4732-3p. We tested the compared the capacity of a synthetic miR-4732-3p mimic with EVs to confer protection to cardiomyocytes, fibroblasts and endothelial cells against oxygen-glucose deprivation (OGD). Results showed that OGD-induced cardiomyocytes treated with either EVs or miR-4732-3p showed prolonged spontaneous beating, lowered ROS levels, and less apoptosis. Transfection of the miR-4732-3p mimic was more effective than EVs in stimulating angiogenesis in vitro and in vivo and in reducing fibroblast differentiation upon transforming growth factor beta treatment. Finally, the miR-4732-3p mimic reduced scar tissue and preserved cardiac function when transplanted intramyocardially in infarcted nude rats. Overall, these results indicate that miR-4732-3p is regulated by hypoxia and exerts cardioprotective actions against ischemic insult, with potential application in cell-free-based therapeutic strategies.

Freshly Thawed Cryobanked Human Neural Stem Cells Engraft within Endogenous Neurogenic Niches and Restore Cognitive Function after Chronic Traumatic Brain Injury.

Human neural stem cells (hNSCs) have potential as a cell therapy after traumatic brain injury (TBI). While various studies have demonstrated the efficacy of NSCs from ongoing culture, there is a significant gap in our understanding of freshly thawed cells from cryobanked stocks-a more clinically relevant source. To address these shortfalls, the therapeutic potential of our previously validated Shef-6.0 human embryonic stem cell (hESC)-derived hNSC line was tested after long-term cryostorage and thawing before transplant. Immunodeficient athymic nude rats received a moderate unilateral controlled cortical impact (CCI) injury. At four weeks post-injury, 6 × 105 freshly thawed hNSCs were transplanted into six injection sites (two ipsi- and four contra-lateral) with 53.4% of cells surviving three months post-transplant. Interestingly, most hNSCs were engrafted in the meninges and the lining of lateral ventricles, associated with high CXCR4 expression and a chemotactic response to SDF1alpha (CXCL12). While some expressed markers of neuron, astrocyte, and oligodendrocyte lineages, the majority remained progenitors, identified through doublecortin expression (78.1%). Importantly, transplantation resulted in improved spatial learning and memory in Morris water maze navigation and reduced risk taking in an elevated plus maze. Investigating potential mechanisms of action, we identified an increase in ipsilateral host hippocampus cornu ammonis (CA) neuron survival, contralateral dentate gyrus (DG) volume, and DG neural progenitor morphology as well as a reduction in neuroinflammation. Together, these findings validate the potential of hNSCs to improve function after TBI and demonstrate that long-term biobanking of cells and thawing aliquots before use may be suitable for clinical deployment.

SHP-1/STAT3 Interaction Is Related to Luteolin-Induced Myocardial Ischemia Protection.

—Prevention and management of myocardial ischemia/reperfusion (I/R) injury is a key step in coronary heart disease surgery. Luteolin is a falconoid compound that has an antioxidant effect, but its mechanism in I/R injury in vivo and in vitro is still under explored. This study attempted to reveal the role of luteolin (Lut) in I/R through mediation of the Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1)/Signal transducer and activator of transcription 3 (STAT3) pathway. To establish I/R rat models, the left anterior descending artery (LAD) was ligated for 30 min and re-perfused for 1 h in Lut-pretreated or nude rats. Comparisons between infarct area, cardiac dysfunction, and myocardial cell death and inflammatory reaction were performed in I/R-induced rats. Hypoxia/reoxygenation (H/R) cell models were established by stimulating H9c2 cells with 95% nitrogen and 5% carbon dioxide. Simultaneously, H/R-related cell death and inflammatory reactions were investigated following Lut treatment. The target protein of Lut was identified using western blotting. Pro-inflammatory cytokines were also measured in serum or Lut-pretreated cell culture medium. The results revealed that compared with the I/R group, Lut treatment could significantly decrease myocardial infarction (MI) area, increase left ventricular ejection fraction (LVEF), and decrease cell death and pro-inflammatory cytokines in the serum. Decreased apoptosis and inflammatory cytokines were also observed in H/R cells after Lut treatment. Lut treatment downregulated SHP-1 expression and subsequently upregulated STAT3 phosphorylation in both I/R rat heart tissue and H9c2 cells. The findings of the current study suggest that Lut can protect the heart and reduce MI area, cell apoptosis rate, and inflammatory level in I/R models.

Engineered neural tissue made using clinical-grade human neural stem cells supports regeneration in a long gap peripheral nerve injury model

A surgical autograft remains the clinical gold-standard therapy for gap repair following peripheral nerve injury, however, challenges remain with achieving full recovery and reducing donor-site morbidity. Engineered Neural Tissue (EngNT) manufactured using differentiated CTX0E03 human stem cells (EngNT-CTX) has been developed as a potential ‘off the shelf’ allogeneic autograft replacement. Ensheathed within a collagen membrane developed to facilitate biomechanical integration, EngNT-CTX was used to bridge a critical-length (15 mm) sciatic nerve gap injury in athymic nude rats. The effectiveness of EngNT-CTX was compared to an autograft using outcome measures that assessed neuronal regeneration and functional recovery at 8 and 16 weeks. At both time points EngNT-CTX restored electrophysiological nerve conduction and functional reinnervation of downstream muscles to the same extent as the autograft. Histological analysis confirmed that more motor neurons had successfully regenerated through the repair in EngNT-CTX in comparison to the autograft at 8 weeks, which was consistent with the electrophysiology, with the number of motor neurons similar in both groups by 16 weeks. The total number of neurons (motor + sensory) was greater in autografts than EngNT-CTX at 8 weeks, indicating that more sensory fibres may have sprouted in those animals at this time point. In conclusion, this study provides evidence to support the effectiveness of EngNT-CTX as a replacement for the nerve autograft, as the functional regeneration assessed through histological and electrophysiological outcome measures demonstrated equivalent performance. Statement of significanceFollowing injury a peripheral nerve has the capacity to regenerate naturally, however, in the case of severe damage where there is a gap the current gold-standard microsurgical intervention is an autograft. This is associated with serious limitations including tissue availability and donor-site morbidity.Tissue engineering aims to overcome these limitations by building a construct from therapeutic cells and biomaterials as a means to mimic and replace the autograft. In this study engineered neural tissue (EngNT) was manufactured using human stem cells (CTX) to bridge a critical-length gap injury. When compared to the autograft in an animal model the EngNT-CTX construct restored function to an equivalent or greater extent.

Changes in Phospholipid/Ceramide Profiles and Eicosanoid Levels in the Plasma of Rats Irradiated with UV Rays and Treated Topically with Cannabidiol.

Chronic UV radiation causes oxidative stress and inflammation of skin and blood cells. Therefore, in this study, we assessed the effects of cannabidiol (CBD), a natural phytocannabinoid with antioxidant and anti-inflammatory properties, on the phospholipid (PL) and ceramide (CER) profiles in the plasma of nude rats irradiated with UVA/UVB and treated topically with CBD. The results obtained showed that UVA/UVB radiation increased the levels of phosphatidylcholines, lysophospholipids, and eicosanoids (PGE2, TxB2), while downregulation of sphingomyelins led to an increase in CER[NS] and CER[NDS]. Topical application of CBD to the skin of control rats significantly upregulated plasma ether-linked phosphatidylethanolamines (PEo) and ceramides. However, CBD administered to rats irradiated with UVA/UVB promoted further upregulation of CER and PEo and led to significant downregulation of lysophospholipids. This was accompanied by the anti-inflammatory effect of CBD, manifested by a reduction in the levels of proinflammatory PGE2 and TxB2 and a dramatic increase in the level of anti-inflammatory LPXA4. It can therefore be suggested that topical application of CBD to the skin of rats exposed to UVA/UVB radiation prevents changes in plasma phospholipid profile resulting in a reduction of inflammation by reducing the level of LPE and LPC species and increasing antioxidant capacity due to upregulation of PEo species.

Dietary Melatonin and Omega-3 Fatty Acids Induce Human Cancer Xenograft Regression In Vivo in Rats by Suppressing Linoleic Acid Uptake and Metabolism.

Melatonin, the circadian nighttime neurohormone, and eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA), which are omega-3 fatty acids (FA) found in high concentrations in fish oil (FO) and plants, abrogate the oncogenic effects of linoleic acid (LA), an omega-6 FA, on the growth of rodent tumors and human breast, prostate, and head and neck squamous cell carcinoma (HNSCC) xenografts in vivo. Here we determined and compared the long-term effects of these inhibitory agents on tumor regression and LA uptake and metabolism to the mitogenic agent 13-[S]-hydroxyoctadecadienoic acid (13-[S]-HODE) in human prostate cancer 3 (PC3) and FaDu HNSCC xenografts in tumor-bearing male nude rats. Rats in this study were split into 3 groups and fed one of 2 diets: one diet containing 5% corn oil (CO, high LA), 5% CO oil and melatonin (2 μg/mL) or an alternative diet 5% FO (low LA). Rats whose diet contained melatonin had a faster rate of regression of PC3 prostate cancer xenografts than those receiving the FO diet, while both in the melatonin and FO groups induced the same rate of regression of HNSCC xenografts. The results also demonstrated that dietary intake of melatonin or FO significantly inhibited tumor LA uptake, cAMP content, 13-[S]-HODE formation, [³H]-thymidine incorporation into tumor DNA, and tumor DNA content. Therefore, long-term ingestion of either melatonin or FO can induce regression of PC3 prostate and HNSCC xenografts via a mechanism involving the suppression of LA uptake and metabolism by the tumor cells.

A therapeutic vascular conduit to support in vivo cell-secreted therapy

A significant barrier to implementation of cell-based therapies is providing adequate vascularization to provide oxygen and nutrients. Here we describe an approach for cell transplantation termed the Therapeutic Vascular Conduit (TVC), which uses an acellular vessel as a scaffold for a hydrogel sheath containing cells designed to secrete a therapeutic protein. The TVC can be directly anastomosed as a vascular graft. Modeling supports the concept that the TVC allows oxygenated blood to flow in close proximity to the transplanted cells to prevent hypoxia. As a proof-of-principle study, we used erythropoietin (EPO) as a model therapeutic protein. If implanted as an arteriovenous vascular graft, such a construct could serve a dual role as an EPO delivery platform and hemodialysis access for patients with end-stage renal disease. When implanted into nude rats, TVCs containing EPO-secreting fibroblasts were able to increase serum EPO and hemoglobin levels for up to 4 weeks. However, constitutive EPO expression resulted in macrophage infiltration and luminal obstruction of the TVC, thus limiting longer-term efficacy. Follow-up in vitro studies support the hypothesis that EPO also functions to recruit macrophages. The TVC is a promising approach to cell-based therapeutic delivery that has the potential to overcome the oxygenation barrier to large-scale cellular implantation and could thus be used for a myriad of clinical disorders. However, a complete understanding of the biological effects of the selected therapeutic is absolutely essential.

6: Creating Tissue Engineered Nipples Using 3d-printed Poly-4-hydroxybutyrate (p4hb) Bioabsorbable Scaffolds Augmented With Autologous Processed Costal Cartilage

Purpose:Nipple reconstruction is a vital part of breast reconstruction after total mastectomy. However, nearly all autologous tissue techniques utilized for reconstruction of the nipple are hindered by scar contracture and loss of projection of the neo-nipple. Unfortunately, engineered tissue substitutes, such as Cook Biodesign® nipple reconstruction cylinders, have demonstrated suboptimal projection maintenance over time. Costal cartilage (CC) has been described in nipple reconstruction to maintain projection, yet has not been adopted due to the excessively firm nipple that results. Herein we propose using 3D-printed bioabsorbable P4HB (3D-P4HB) scaffolds loaded with processed CC in order to foster ingrowth of tissue that mimics the biomechanical properties of native nipples and to protect the regenerated tissue from contracture as it matures.Methods:3D-P4HB scaffolds (diameter: 1.0cm, height: 1.0cm) were fabricated and sterilized. Patient-derived CC (discarded from DIEP procedures) was either minced (1mm3) or zested (<0.2mm3) in sterile fashion. Processed cartilage-filled 3D-P4HB scaffolds were subcutaneously implanted into nude rats using a CV flap technique. Additional groups consisted of empty 3D-P4HB scaffolds, 3D-P4HB scaffolds with an internal 3D latticework of P4HB filaments (rebar), and non-scaffolded (naked) cartilage. The constructs were explanted at 1, 3 and 6 months, and evaluated by gross, microstructural, histological, and biomechanical analysis.Results:All 3D-P4HB nipple reconstructions were well preserved in diameter and projection at 1, 3 and 6 months, primarily due to the persistence of the scaffold architecture throughout these time points. When compared to the non-scaffolded (naked) group, 3D-P4HB groups demonstrated significantly greater projection at 3 and 6 months (p<0.05). There were no significant differences observed in tissue volume retention between the two processed cartilage-filled 3D-P4HB groups at 1, 3 and 6 months. However, the non-scaffolded (naked) group lost a significant amount of volume in the first 3 months (38% in minced and 26% in zested, p<0.05), but remained unchanged between 3 and 6 months. Newly formed spongy fibrovascular cartilaginous tissue (with viable chondrocytes within the lacunae) was also noted in processed cartilage-filled 3D-P4HB groups. Interestingly, 3D-P4HB (rebar) scaffolds demonstrated the fastest material absorption overtime, as demonstrated by biomechanical testing and SEM analysis which verified widespread pitting on the material surface. Elastic modulus testing indicated minimal change in 3D-P4HB (rebar) scaffold stiffness at 1 month, yet a sharp decrease from 8MPa to 4MPa between 1 to 3 months. Both processed cartilage-filled 3D-P4HB scaffolds slightly increased in stiffness over 6 months within the range of 2 to 3MPa (p>0.05).Conclusion:Using 3D-P4HB scaffolds filled with autologous processed CC, we have engineered nipples that maintain their projection and volume over time, while simultaneously allowing for the maturation of an internal structure of fibrovascular cartilaginous tissue that biomechanically mimics that of native nipples. As the 3D-P4HB scaffold gradually absorbs, data suggest that newly-regenerated tissue may resist scar contracture and maintain projection over time. Because P4HB devices for soft tissue reinforcement have previously been cleared by the FDA and possess a long track record of safety, we believe that this novel 3D-P4HB nipple reconstruction scaffold may be readily translatable to the clinic.