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Novel strategies have been proposed for articular cartilage damage occurring during osteoarthritis (OA) and -among these- Extracorporeal Shock Wave Therapy (ESWT), intra-articular injections of Platelet-Rich Plasma (PRP) or Hyaluronic Acid (HA) revealed encouraging results. To investigate the possible mechanisms responsible for those clinical benefits, we established primary cultures of human chondrocytes derived from cartilage explants and measured the in vitro effects of ESW, PRP and HA therapies. After molecular/morphological cell characterization, we assessed those effects on the functional activities of the chondrocyte cell cultures, at the protein and molecular levels. ESWT significantly prevented the progressive dedifferentiation that spontaneously occurs during prolonged chondrocyte culture. We then attested the efficiency of all such treatments to stimulate the expression of markers of chondrogenic potential such as SOX9 and COL2A, to increase the Ki67 proliferation index as well as to antagonize the traditional marker of chondrosenescence p16INK4a (known as Cdkn2a). Furthermore, all our samples showed an ESW- and HA-mediated enhancement of migratory and anti-inflammatory activity onto the cytokine-rich environment characterizing OA. Taken together, those results suggest a regenerative effect of such therapies on primary human chondrocytes in vitro. Moreover, we also show for the first time that ESW treatment induces the surface expression of major hyaluronan cell receptor CD44 allowing the increase of COL2A/COL1A ratio upon HA administration. Therefore, this work suggests that ESW-induced CD44 overexpression enhances the in vitro cell susceptibility of human chondrocytes to HA, presumably favouring the repair of degenerated cartilage. [ABSTRACT FROM AUTHOR]

Purpose: We have previously reported that lamellar dissection of the cornea transects stromal nerves, and that regenerating neurites form a dense net along the surgical plane. In these experiments, we have disrupted the stromal nerve trunks in situ, without incising the cornea, to determine the regeneration events in the absence of a surgical plane. Methods: Thy1-YFP mice were anesthetized and in vivo images of the corneal nerves were obtained with a wide-field stereofluorescent microscope. A far infrared XYRCOS Laser attached to 20X objective of an upright microscope was used to perform in situ transection of the stromal nerves. 3 types of laser transections were performed (n = 5/group): (i) point transection (a single cut); (ii) segmental transection (two cuts enclosing a segment of nerve trunk); and (iii) annular transection (cuts on all nerve trunks crossing the perimeter of a 0.8 mm diameter circular area centered on the corneal apex). Mice were imaged sequentially for 4 weeks thereafter to assess nerve degeneration (disappearance or weakening of original fluorescence intensity) or regeneration (appearance of new fluorescent fronds). Beta-3-tubulin immunostaining was performed on corneal whole-mounts to demonstrate nerve disruption. Results: The pattern of stromal nerves in corneas of the same mouse and in corneas of littermates was dissimilar. Two distinct patterns were observed, often within the same cornea: (i) interconnected trunks that spanned limbus to limbus; or (ii) dichotomously branching trunks that terminate at the corneal apex. Point transections did not cause degeneration of proximal or distal segment in interconnected trunks, but resulted in degeneration of distal segment of branching trunks. In segmental transections, the nerve segment enclosed within the two laser cuts degenerated. Lack of beta-3 tubulin staining at transection site confirmed nerve transection. In interconnected trunks, at 4 weeks, a hyperfluorescent plaque filled the gap created by the transection. In annular transections, some nerve trunks degenerated, while others regained or retained fluorescence. Conclusions: Interconnected stromal nerves in murine corneas do not degenerate after in situ point transection and show evidence of healing at the site of disruption. Presence or absence of a surgical plane influences corneal nerve regeneration after transection. [ABSTRACT FROM AUTHOR]

Human platelet lysate (hPL) has been considered as the preferred supplement for the xeno-free stem cell culture for many years. However, the biological effect of hPL on the proliferation and differentiation of dental stem cells combined with the use of medical grade synthetic biomaterial is still under investigation. Thus, the optimal scaffold composition, cell type and specific growth conditions, yet need to be formulated. In this study, we aimed to investigate the regenerative potential of dental stem cells seeded on synthetic scaffolds and maintained in osteogenic media supplemented with either hPL or xeno-derived fetal bovine serum (FBS). Two types of dental stem cells were isolated from human impacted third molars and intact teeth; stem cells of apical papilla (SCAP) and periodontal ligament stem cells (PDLSCs). Cells were expanded in cell culture media supplemented with either hPL or FBS. Consequently, proliferative capacity, immunophenotypic characteristics and multilineage differentiation potential of the derived cells were evaluated on monolayer culture (2D) and on synthetic scaffolds fabricated from poly ’lactic-co-glycolic’ acid (PLGA) (3D). The functionality of the induced cells was examined by measuring the concentration of osteogenic markers ALP, OCN and OPN at different time points. Our results indicate that the isolated dental stem cells showed similar mesenchymal characteristics when cultured on hPL or FBS-containing culture media. Scanning electron microscopy (SEM) and H&E staining revealed the proper adherence of the derived cells on the 3D scaffold cultures. Moreover, the increase in the concentration of osteogenic markers proved that hPL was able to produce functional osteoblasts in both culture conditions (2D and 3D), in a way similar to FBS culture. These results reveal that hPL provides a suitable substitute to the animal-derived serum, for the growth and functionality of both SCAP and PDLSCs. Thus the use of hPL, in combination with PLGA scaffolds, can be useful in future clinical trials for dental regeneration. [ABSTRACT FROM AUTHOR]

Many lung diseases, such as the acute respiratory distress syndrome (ARDS), display significant regional heterogeneity with patches of severely injured tissue adjacent to apparently healthy tissue. Current mouse models that aim to mimic ARDS generally produce diffuse injuries that cannot reproducibly generate ARDS’s regional heterogeneity. This deficiency prevents the evaluation of how well therapeutic agents reach the most injured regions and precludes many regenerative medicine studies since it is not possible to know which apparently healing regions suffered severe injury initially. Finally, these diffuse injury models must be relatively mild to allow for survival, as their diffuse nature does not allow for residual healthy lung to keep an animal alive long enough for many drug and regenerative medicine studies. To solve all of these deficiencies in current animal models, we have created a simple and reproducible technique to selectively induce lung injury in specific areas of the lung. Our technique, catheter-in-catheter selective lung injury (CICSLI), involves guiding an inner catheter to a particular area of the lung and delivering an injurious agent mixed with nanoparticles (fluorescently and/or radioactively labeled) that can be used days later to track the location and extent of where the initial injury occurred. Furthermore, we demonstrate that CICSLI can produce a more severe injury than diffuse models, yet has much higher survival since CICSLI intentionally leaves lung regions undamaged. Collectively, these attributes of CICSLI will allow investigators to better study how drugs act within heterogeneous lung pathologies and how regeneration occurs in severely damaged lung tissue, thereby aiding the development of new therapies for ARDS and other heterogenous lung diseases. [ABSTRACT FROM AUTHOR]

Metabolic syndrome (MS), overlapping type 2 diabetes, hyperlipidemia, and/or hypertension, owing to high-fat diet, poses risk for cardiovascular disease. A critical feature associated with such risk is the functional impairment of endothelial progenitor cells (EPCs). Dipeptidyl dipeptidase-4 inhibitors (DPP-4 i) not only inhibit degradation of incretins to control blood glucose levels, but also improve EPC bioactivity and induce anti-inflammatory effects in tissues. In the present study, we investigated the effects of such an inhibitor, MK-06266, in an ischemia model of MS using diet-induced obese (DIO) mice. EPC bioactivity was examined in MK-0626-administered DIO mice and a non-treated control group, using an EPC colony-forming assay and bone marrow cKit+ Sca-1+ lineage-cells, and peripheral blood-mononuclear cells. Our results showed that, in vitro, the effect of MK-0626 treatment on EPC bioactivities and differentiation was superior compared to the control. Furthermore, microvascular density and pericyte-recruited arteriole number increased in MK-0626-administered mice, but not in the control group. Lineage profiling of isolated cells from ischemic tissues revealed that MK-0626 administration has an inhibitory effect on unproductive inflammation. This occurred via a decrease in the influx of total blood cells and pro-inflammatory cells such as neutrophils, total macrophages, M1, total T-cells, cytotoxic T-cells, and B-cells, with a concomitant increase in number of regeneration-associated cells, such as M2/M ratio and Treg/T-helper. Laser Doppler analysis revealed that at day 14 after ischemic injury, blood perfusion in hindlimb was greater in MK-0626-treated DIO mice, but not in control. In conclusion, the DPP-4 i had a positive effect on EPC differentiation in MS model of DIO mice. Following ischemic injury, DPP-4 i sharply reduced recruitment of pro-inflammatory cells into ischemic tissue and triggered regeneration and reparation, making it a promising therapeutic agent for MS treatment. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) can trans/differentiate to neural precursors and/or mature neurons and promote neuroprotection and neurogenesis. The above could greatly benefit neurodegenerative disorders as well as in the treatment of post-traumatic and hereditary diseases of the central nervous system (CNS). In order to attain an ideal source of adult MSCs for the treatment of CNS diseases, adipose tissue, bone marrow, skin and umbilical cord derived MSCs were isolated and studied to explore differences with regard to neural differentiation capacity. In this study, we demonstrated that MSCs from several tissues can differentiate into neuron-like cells and differentially express progenitors and mature neural markers. Adipose tissue MSCs exhibited significantly higher expression of neural markers and had a faster proliferation rate. Our results suggest that adipose tissue MSCs are the best candidates for the use in neurological diseases. [ABSTRACT FROM AUTHOR]

It has been reported that donor age affects patient outcomes after liver transplantation, and that telomere length is associated with age. However, to our knowledge, the impact of donor age and donor liver telomere length in liver transplantation has not been well investigated. This study aimed to clarify the influence of the length of telomere and G-tail from donor livers on the outcomes of living donors and recipients after living donor liver transplantation. The length of telomere and G-tail derived from blood samples and liver tissues of 55 living donors, measured using the hybridization protection assay. The length of telomeres from blood samples was inversely correlated with ages, whereas G-tail length from blood samples and telomere and G-tail lengths from liver tissues were not correlated with ages. Age, telomere, and G-tail length from blood did not affect postoperative liver failure and early liver regeneration of donors. On the other hand, the longer the liver telomere, the poorer the liver regeneration tended to be, especially with significant difference in donor who underwent right hemihepatectomy. We found that the survival rate of recipients who received liver graft with longer telomeres was inferior to that of those who received liver graft with shorter ones. An elderly donor, longer liver telomere, and higher Model for End-Stage Liver Disease score were identified as independent risk factors for recipient survival after transplantation. In conclusion, telomere shortening in healthy liver does not correlate with age, whereas longer liver telomeres negatively influence donor liver regeneration and recipient survival after living donor liver transplantation. These results can direct future studies and investigations on telomere shortening in the clinical and experimental transplant setting. [ABSTRACT FROM AUTHOR]

The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-eGFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch’s membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo. [ABSTRACT FROM AUTHOR]

Background: Considering the impaired function of regenerative cells in myocardial infarction (MI) patients with comorbidities and associated risk factors, cell therapy to enhance the regenerative microenvironment was designed using regeneration-associated cells (RACs), including endothelial progenitor cells (EPCs) and anti-inflammatory cells. Methods: RACs were prepared by quality and quantity control culture of blood mononuclear cells (QQMNCs). Peripheral blood mononuclear cells (PBMNCs) were isolated from Lewis rats and conditioned for 5 days using a medium containing stem cell factors, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6 to generate QQMNCs. Results: A 5.3-fold increase in the definitive colony-forming EPCs and vasculogenic EPCs was observed, in comparison to naïve PBMNCs. QQMNCs were enriched with EPCs (28.9-fold, P<0.0019) and M2 macrophages (160.3-fold, P<0.0002). Genes involved in angiogenesis (angpt1, angpt2, and vegfb), stem/progenitors (c-kit and sca-1), and anti-inflammation (arg-1, erg-2, tgfb, and foxp3) were upregulated in QQMNCs. For in vivo experiments, cells were administered into syngeneic rat models of MI. QQMNC-transplanted group (QQ-Tx) preserved cardiac function and fraction shortening 28 days post-MI in comparison with PBMNCs-transplanted (PB-Tx) (P<0.0001) and Control (P<0.0008) groups. QQ-Tx showed enhanced angiogenesis and reduced interstitial left ventricular fibrosis, along with a decrease in neutrophils and an increase in M2 macrophages in the acute phase of MI. Cell tracing studies revealed that intravenously administered QQMNCs preferentially homed to ischemic tissues via blood circulation. QQ-Tx showed markedly upregulated early cardiac transcriptional cofactors (Nkx2-5, 29.8-fold, and Gata-4, 5.2-fold) as well as c-kit (4.5-fold) while these markers were downregulated in PB-Tx. In QQ-Tx animals, de novo blood vessels formed a “Biological Bypass”, observed macroscopically and microscopically, while PB-Tx and Control-Tx groups showed severe fibrotic adhesion to the surrounding tissues, but no epicardial blood vessels. Conclusion: QQMNCs conferred potent angiogenic and anti-inflammatory properties to the regenerative microenvironment, enhancing myocardiogenesis and functional recovery of rat MI hearts. [ABSTRACT FROM AUTHOR]

Cancer treatments including ionizing radiation (IR) can induce cancer stem cell-like properties in non-stem cancer cells, an outcome that can interfere with therapeutic success. Yet, we understand little about what consequences of IR induces stem cell like properties and why some cancer cells show this response but not others. In previous studies, we identified a pool of epithelial cells in Drosophila larval wing discs that display IR-induced stem cell-like properties. These cells are resistant to killing by IR and, after radiation damage, change fate and translocate to regenerate parts of the disc that suffered more cell death. Here, we report the identification of two new pools of cells with IR-induced regenerative capability. We addressed how IR exposure results in the induction of stem cell-like behavior, and found a requirement for IR-induced caspase activity and for Zfh2, a transcription factor and an effector in the JAK/STAT pathway. Unexpectedly, the requirement for caspase activity was cell-autonomous within cell populations that display regenerative behavior. We propose a model in which the requirement for caspase activity and Zfh2 can be explained by apoptotic and non-apoptotic functions of caspases in the induction of stem cell-like behavior. [ABSTRACT FROM AUTHOR]

Necroptosis is a programmed necrosis, regulated by receptor interacting protein kinase 1(RIP1) and receptor interacting protein kinase 3(RIP3), and could be inhibited by necrostatin-1(Nec-1) specifically. This study aims to evaluate the effect of Nec-1 on LPS-treated periodontal ligament stem cells (PDLSCs). In the research, three groups were established: normal cultured PDLSCs, Porphyromonas gingivalis (Pg)-LPS stimulated PDLSCs and Pg-LPS+Nec-1 treated PDLSCs. The expression of RIP1 and RIP3 and osteogenic differentiation of PDLSCs in three groups were analyzed. Then, we constructed cell aggregates (CA) using PDLSCs, then PDLSCs-CA were combined with Bio-Oss in three groups were transplanted subcutaneously in nude mice to assess their potentials of periodontal tissue regeneration. The results showed that RIP1 and RIP3 were fully expressed in Pg-LPS stimulated PDLSCs and the level increased significantly. Nec-1 inhibited RIP1-RIP3 interaction, and further inhibited necroptosis of PDLSCs in inflammatory state. Moreover, Nec-1 pretreatment ameliorates the osteogenic differentiation of LPS-treated PDLSCs and can effectively promote the cementum like structure ectopic regenerative ability of PDLSCs in nude mice. These findings show RIP1/RIP3-mediated necroptosis is an important mechanism of cell death in PDLSCs. Nec-1 has a protective effect in reducing cell death and promotes ectopic periodontal tissue like structure regeneration by inhibiting necroptosis. Nec-1 is a hopeful therapeutic agent which protects cells from necroptosis and ameliorates functional outcome. [ABSTRACT FROM AUTHOR]

Mice lacking the sarcolemmal protein dystrophin, designated mdx, have been widely used as a model of Duchenne muscular dystrophy. Dystrophic mdx mice as they mature develop notable morphological abnormalities to their neuromuscular junctions, the peripheral cholinergic synapses responsible for activating muscle fibers. Most obviously the acetylcholine receptor aggregates are fragmented into small non-continuous, islands. This contrasts with wild type mice whose acetylcholine receptor aggregates are continuous and pretzel-shaped in appearance. We show here that these abnormalities in mdx mice are also present in a canine model of Duchenne muscular dystrophy and provide additional evidence to support the hypothesis that NMJ remodeling occurs due to myofiber degeneration and regeneration. Using a method to investigate synaptic AChR replacement, we show that neuromuscular junction remodeling in mdx animals is caused by muscle fiber degeneration and regeneration at the synaptic site and is mimicked by deliberate myofiber injury in wild type mice. Importantly, the innervating motor axon plays a crucial role in directing the remodeling of the neuromuscular junction in dystrophy, as has been recorded in aging and deliberate muscle fiber injury in wild type mice. The remodeling occurs repetitively through the life of the animal and the changes in junctions become greater with age. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) followed by extensive cell loss, inflammation, and scarring, often permanently damages neurological function. Biomaterial scaffolds are promising but currently have limited applicability in SCI because after entering the scaffold, regenerating axons tend to become trapped and rarelyre-enter the host tissue, the reasons for which remain to be completely explored. Here, we propose a mathematical model and computer simulation for characterizing regenerative axons growing along a scaffold following SCI, and how their growth may be guided. The model assumed a solid, spherical, multifunctional, biomaterial scaffold, that would bridge the rostral and caudal stumps of a completely transected spinal cord in a rat model and would guide the rostral regenerative axons toward the caudal tissue. Other assumptions include the whole scaffold being coated with extracellular matrix components, and the caudal area being additionally seeded with chemoattractants. The chemical factors on and around the scaffold were formulated to several coupled variables, and the parameter values were derived fromexisting experimental data. Special attention was given to the effects of coating strength, seeding location, and seeding density, as well as the ramp slope of the scaffold, on axonal regeneration. In numerical simulations, a slimmer scaffold provided a small slope at the entry “on-ramp” area that improved the success rate of axonal regeneration. If success rates are high, an increased number of regenerative axons traverse through the narrow channels, causing congestion and lowering the growth rate. An increase in the number of severed axons (300–12000) did not significantly affect the growth rate, but it reduced the success rate of axonal regeneration. However, an increase in the seeding densities of the complexes on the whole scaffold, and that in the seeding densities of the chemoattractants on the caudal area, improved both the success and growth rates. However, an increase in the density of thecomplexes on the whole scaffold risks an over-eutrophic surface that harms axonal regeneration.Although theoretical predictions are yet to be validated directly by experiments, this theoretical tool can advance the treatment of SCI, and is also applicable to scaffolds with other architectures. [ABSTRACT FROM AUTHOR]

Objectives: We investigated whether chronological changes in portal flow and clinical factors play a role in the liver regeneration (LR) process after right donor-hepatectomy. Materials and methods: Participants in this prospective study comprised 58 donors who underwent right donor-hepatectomy during the period February 2014 to February 2015 at a single medical institution. LR was estimated using two equations: remnant left liver (RLL) growth (%) and liver volumetric recovery (LVR) (%). Donors were classified into an excellent regeneration (ER) group or a moderate regeneration (MR) group based on how their LR on postoperative day 7 compared to the median value. Results: Multivariate analysis revealed that low residual liver volume (OR = .569, 95% CI: .367– .882) and high portal venous velocity in the immediate postoperative period (OR = 1.220, 95% CI: 1.001–1.488) were significant predictors of LR using the RLL growth equation; high portal venous velocity in the immediate postoperative period (OR = 1.325, 95% CI: 1.081–1.622) was a significant predictor of LR using the LVR equation. Based on the two equations, long-term LR was significantly greater in the ER group than in the MR group (p < .001). Conclusion: Portal venous velocity in the immediate postoperative period was an important factor in LR. The critical time for short-term LR is postoperative day 7; it is associated with long-term LR in donor-hepatectomy. [ABSTRACT FROM AUTHOR]

Objective: We sought to define the intrinsic stem cell capacity in pediatric heart lesions, and the effects of diagnosis and of age, in order to inform evidence-based use of potential autologous stem cell sources for regenerative medicine therapy. Methods: Ventricular explants derived from patients with hypoplastic left heart syndrome (HLHS), tetralogy of Fallot (TF), dilated cardiomyopathy (DCM) and ventricular septal defect (VSD) were analyzed following standard in vitro culture conditions, which yielded cardiospheres (C-spheres), indicative of endogenous stem cell capacity. C-sphere counts generated per 5 mm3 tissue explant and the presence of cardiac progenitor cells were correlated to patient age, diagnosis and echocardiographic function. Results: Cardiac explants from patients less than one year of age with TF and DCM robustly generated c-kit- and/or vimentin-positive cardiac mesenchymal cells (CMCs), populating spontaneously forming C-spheres. Beyond one year of age, there was a marked reduction or absence of cardiac explant-derivable cardiac stem cell content in patients with TF, VSD and DCM. Stem cell content in HLHS and DCM strongly correlated to the echocardiographic function in the corresponding ventricular chamber, with better echocardiographic function correlating to a more robust regenerative cellular content. Conclusions: We conclude that autologous cardiomyogenic potential in pediatric heart lesions is robust during the first year of life and uniformly declines thereafter. Depletion of stem cell content occurs at an earlier age in HLHS with the onset of ventricular failure in a chamber-specific pattern that correlates directly to ventricular dysfunction. These data suggest that regenerative therapies using autologous cellular sources should be implemented in the neonatal period before the potentially rapid onset of single ventricle failure in HLHS or the evolution of biventricular failure in DCM. [ABSTRACT FROM AUTHOR]

This study was designed to characterize morphologic stages during neuroma development post amputation with an eye toward developing better treatment strategies that intervene before neuromas are fully formed. Right forelimbs of 30 Sprague Dawley rats were amputated and limb stumps were collected at 3, 7, 28, 60 and 90 ays ost mputation (DPA). Morphology of newly formed nerves and neuromas were assessed via general histology and neurofilament protein antibody staining. Analysis revealed six morphological characteristics during nerve and neuroma development; 1) normal nerve, 2) degenerating axons, 3) axonal sprouts, 4) unorganized bundles of axons, 5) unorganized axon growth into muscles, and 6) unorganized axon growth into fibrotic tissue (neuroma). At early stages (3 & 7 DPA) after amputation, normal nerves could be identified throughout the limb stump and small areas of axonal sprouts were present near the site of injury. Signs of degenerating axons were evident from 7 to 90 DPA. From day 28 on, variability of nerve characteristics with signs of unorganized axon growth into muscle and fibrotic tissue and neuroma formation became visible in multiple areas of stump tissue. These pathological features became more evident on days 60 and 90. At 90 DPA frank neuroma formation was present in all stump tissue. By following nerve regrowth and neuroma formation after amputation we were able to identify 6 separate histological stages of nerve regrowth and neuroma development. Axonal regrowth was observed as early as 3 DPA and signs of unorganized axonal growth and neuroma formation were evident by 28 DPA. Based on these observations we speculate that neuroma treatment and or prevention strategies might be more successful if targeted at the initial stages of development and not after 28 DPA. [ABSTRACT FROM AUTHOR]

Endothelial cells (EC) frequently undergo primary or secondary injury during kidney disease such as thrombotic microangiopathy or glomerulonephritis. Renin Lineage Cells (RLCs) serve as a progenitor cell niche after glomerular damage in the adult kidney. However, it is not clear whether RLCs also contribute to endothelial replenishment in the glomerulus following endothelial injury. Therefore, we investigated the role of RLCs as a potential progenitor niche for glomerular endothelial regeneration. We used an inducible tet-on triple-transgenic reporter strain mRen-rtTAm2/LC1/LacZ to pulse-label the renin-producing RLCs in adult mice. Unilateral kidney EC damage (EC model) was induced by renal artery perfusion with concanavalin/anti-concanavalin. In this model glomerular EC injury and depletion developed within 1 day while regeneration occurred after 7 days. LacZ-labelled RLCs were restricted to the juxtaglomerular compartment of the afferent arterioles at baseline conditions. In contrast, during the regenerative phase of the EC model (day 7) a subset of LacZ-tagged RLCs migrated to the glomerular tuft. Intraglomerular RLCs did not express renin anymore and did not stain for glomerular endothelial or podocyte cell markers, but for the mesangial cell markers α8-integrin and PDGFRβ. Accordingly, we found pronounced mesangial cell damage parallel to the endothelial injury induced by the EC model. These results demonstrated that in our EC model RLCs are not involved in endothelial regeneration. Rather, recruitment of RLCs seems to be specific for the repair of the concomitantly damaged mesangium. [ABSTRACT FROM AUTHOR]

A variety of conditions ranging from glaucoma to blunt force trauma lead to optic nerve atrophy. Identifying signaling pathways for stimulating axon growth in the optic nerve may lead to treatments for these pathologies. Inhibiting signaling by the nogo-66 receptor 1 (NgR1) promotes the re-extension of axons following a crush injury to the optic nerve, and while NgR1 mRNA and protein expression are observed in the retinal ganglion cell (RGC) layer and inner nuclear layer, which retinal cell types express NgR1 remains unknown. Here we determine the expression pattern of NgR1 in the mouse retina by co-labeling neurons with characterized markers of specific retinal neurons together with antibodies specific for NgR1 or Green Fluorescent Protein expressed under control of the ngr1 promoter. We demonstrate that more than 99% of RGCs express NgR1. Thus, inhibiting NgR1 function may ubiquitously promote the regeneration of axons by RGCs. These results provide additional support for the therapeutic potential of NgR1 signaling in reversing optic nerve atrophy. [ABSTRACT FROM AUTHOR]

Background: Liver graft regeneration is orchestrated by specific and sequential stimuli, including hepatocyte growth factors, cytokines, and catecholamines. We evaluated the association between preoperative serum cytokines and early liver graft regeneration in human living donor liver transplantation (LDLT). Patients and methods: We retrospectively reviewed the data of adult patients who underwent LDLT from January 2010 to December 2014. Serum cytokines, including interleukin (IL)-2, 6, 10, 12, 17, interferon (IFN)-γ and tumor necrosis factor (TNF)-α were measured in the recipients 1 day before surgery and on postoperative day (POD) 7. Liver graft volume was estimated using abdominal computed tomography images of the donors and recipients. Results: In total, 226 patients were analyzed in this study. Median preoperative levels of serum cytokines were as follows: IL-2, 0.1 (0.1–1.6) pg/mL; IL-6, 7.3 (0.1–30.2) pg/mL; IL-10, 0.5 (0.1–11.0) pg/mL; IL-12, 0.1 (0.1–0.1) pg/mL; IL-17, 2.0 (0.1–16.4) pg/mL; IFN-γ, 3.2 (0.1–16.0) pg/mL; and TNF-α, 9.8 (5.4–17.9) pg/mL. Higher preoperative serum levels of IL-6, IL-10, and TNF-α, dichotomized at the median, were associated with increased relative liver volumes by POD 7. Multivariate analysis revealed that higher levels of serum IL-6 and TNF-α were independently associated with increased graft volume during the first 1 week after LDLT, based on the lower levels of those cytokines. Conclusions: IL-6 and TNF-α were important mediators of the success of early graft regeneration in patients who underwent LDLT. [ABSTRACT FROM AUTHOR]

Worldwide tree mortality as induced by climate change presents a challenge to forest managers. To successfully manage vulnerable forests requires the capacity of regeneration to compensate for losses from tree mortality. We observed rapid regeneration and the growth release of young trees after warming-induced mortality in a David aspen-dominated (Populus davidiana) broad-leaved forest in Inner Mongolia, China, as based on individual tree measurements taken in 2012 and 2015 from a 6-ha permanent plot. Warming and drought stress killed large trees 10–15 m tall with a total number of 2881 trees during 2011–2012, and also thinned the upper crowns. David aspen recruitment increased 2 times during 2012–2015 and resulted in a high transition probability of David aspen replacing the same or other species, whereas the recruitment of Mongolian oak (Quercus mongolica) was much lower: it decreased from 2012 to 2015, indicating that rapid regeneration represented a regrowth phase for David aspen, and not succession to Mongolian oak. Further, we found that the recruitment density increased with canopy openness, thus implying that warming-induced mortality enhanced regeneration. Our results suggest that David aspen has a high regrowth ability to offset individual losses from warming-induced mortality. This important insight has implications for managing this vulnerable forest in the semi-arid region of northern China. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) causes progressive disability in 1 of every 5,000 boys due to the lack of functional dystrophin protein. Despite much advancement in knowledge about DMD disease presentation and progression—attributable in part to studies using mouse and canine models of the disease–current DMD treatments are not equally effective in all patients. There remains, therefore, a need for translational animal models in which novel treatment targets can be identified and evaluated. Golden Retriever muscular dystrophy (GRMD) is a phenotypically and genetically homologous animal model of DMD. As with DMD, speed of disease progression in GRMD varies substantially. However, unlike DMD, all GRMD dogs possess the same causal mutation; therefore genetic modifiers of phenotypic variation are relatively easier to identify. Furthermore, the GRMD dogs used in this study reside within the same colony, reducing the confounding effects of environment on phenotypic variation. To detect modifiers of disease progression, we developed gene expression profiles using RNA sequencing for 9 dogs: 6 GRMD dogs (3 with faster-progressing and 3 with slower-progressing disease, based on quantitative, objective biomarkers) and 3 control dogs from the same colony. All dogs were evaluated at 2 time points: early disease onset (3 months of age) and the point at which GRMD stabilizes (6 months of age) using quantitative, objective biomarkers identified as robust against the effects of relatedness/inbreeding. Across all comparisons, the most differentially expressed genes fell into 3 categories: myogenesis/muscle regeneration, metabolism, and inflammation. Our findings are largely in concordance with DMD and mouse model studies, reinforcing the utility of GRMD as a translational model. Novel findings include the strong up-regulation of chitinase 3-like 1 (CHI3L1) in faster-progressing GRMD dogs, suggesting previously unexplored mechanisms underlie progression speed in GRMD and DMD. In summary, our findings support the utility of RNA sequencing for evaluating potential biomarkers of GRMD progression speed, and are valuable for identifying new avenues of exploration in DMD research. [ABSTRACT FROM AUTHOR]

Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy. [ABSTRACT FROM AUTHOR]

Nematostella vectensis is a member of the phylum Cnidaria, a lineage that includes anemones, corals, hydras, and jellyfishes. This estuarine anemone is an excellent model system for investigating the evolution of stress tolerance because it is easy to collect in its natural habitat and to culture in the laboratory, and it has a sequenced genome. Additionally, there is evidence of local adaptation to environmental stress in different N. vectensis populations, and abundant protein-coding polymorphisms have been identified, including polymorphisms in proteins that are implicated in stress responses. N. vectensis can tolerate a wide range of environmental parameters, and has recently been shown to have substantial intraspecific variation in temperature preference. We investigated whether different clonal lines of anemones also exhibit differential tolerance to oxidative stress. N. vectensis populations are continually exposed to reactive oxygen species (ROS) generated during cellular metabolism and by other environmental factors. Fifteen clonal lines of N. vectensis collected from four different estuaries were exposed to hydrogen peroxide. Pronounced differences in survival and regeneration were apparent between clonal lines collected from Meadowlands, NJ, Baruch, SC, and Kingsport, NS, as well as among 12 clonal lines collected from a single Cape Cod marsh. To our knowledge, this is the first example of intraspecific variability in oxidative stress resistance in cnidarians or in any marine animal. As oxidative stress often accompanies heat stress in marine organisms, resistance to oxidative stress could strongly influence survival in warming oceans. For example, while elevated temperatures trigger bleaching in corals, oxidative stress is thought to be the proximal trigger of bleaching at the cellular level. [ABSTRACT FROM AUTHOR]

The glycosylation of alpha-dystroglycan (α-DG) is crucial in maintaining muscle cell membrane integrity. Dystroglycanopathies are identified by the loss of this glycosylation leading to a breakdown of muscle cell membrane integrity and eventual degeneration. However, a small portion of fibers expressing functionally glycosylated α-DG (F-α-DG) (revertant fibers, RF) have been identified. These fibers are generally small in size, centrally nucleated and linked to regenerating fibers. Examination of different muscles have shown various levels of RFs but it is unclear the extent of which they are present. Here we do a body-wide examination of muscles from the FKRP-P448L mutant mouse for the prevalence of RFs. We have identified great variation in the distribution of RF in different muscles and tissues. Triceps shows a large increase in RFs and together with centrally nucleated fibers whereas the pectoralis shows a reduction in revertant but increase in centrally nucleated fibers from 6 weeks to 6 months of age. We have also identified that the sciatic nerve with near normal levels of F-α-DG in the P448Lneo- mouse with reduced levels in the P448Lneo+ and absent in LARGEmyd. The salivary gland of LARGEmyd mice expresses high levels of F-α-DG. Interestingly the same glands in the P448Lneo-and to a lesser degree in P448Lneo+ also maintain considerable amount of F-α-DG, indicating the non-proliferating epithelial cells have a molecular setting permitting glycosylation. [ABSTRACT FROM AUTHOR]

We propose an agent-based model for the simulation of the autoimmune response in T1D. The model incorporates cell behavior from various rules derived from the current literature and is implemented on a high-performance computing system, which enables the simulation of a significant portion of the islets in the mouse pancreas. Simulation results indicate that the model is able to capture the trends that emerge during the progression of the autoimmunity. The multi-scale nature of the model enables definition of rules or equations that govern cellular or sub-cellular level phenomena and observation of the outcomes at the tissue scale. It is expected that such a model would facilitate in vivo clinical studies through rapid testing of hypotheses and planning of future experiments by providing insight into disease progression at different scales, some of which may not be obtained easily in clinical studies. Furthermore, the modular structure of the model simplifies tasks such as the addition of new cell types, and the definition or modification of different behaviors of the environment and the cells with ease. [ABSTRACT FROM AUTHOR]

In April 2010, Austropuccinia psidii (formerly Puccinia psidii) was detected for the first time in Australia on the central coast of New South Wales. The fungus spread rapidly along the east coast and can now be found infecting vegetation in a range of native forest ecosystems with disease impacts ranging from minor leaf spots to severe shoot and stem blight and tree dieback. Localised extinction of some plant species has been recorded. In 2014, the impact of A. psidii was observed for the first time in a wet sclerophyll site with a rainforest understory, dominated by species of Myrtaceae, in Tallebudgera Valley, south east Queensland, Australia. This study aimed to determine the impact of A. psidii on individual species and species composition. Here we provide quantitative and qualitative evidence on the significant impact A. psidii has in native ecosystems, on a broader range of species than previously reported. Archirhodomyrtus beckleri, Decaspermum humile, Gossia hillii and Rhodamnia maideniana are in serious decline, with significant increases in tree mortality over the period of our study. This research further highlights the potential of this invasive pathogen to negatively impact native ecosystems and biodiversity. [ABSTRACT FROM AUTHOR]

Introduction: Degeneration of the intervertebral disc (IVD) is a frequent cause for back pain in humans and dogs. Link-N stabilizes proteoglycan aggregates in cartilaginous tissues and exerts growth factor-like effects. The human variant of Link-N facilitates IVD regeneration in several species in vitro by inducing Smad1 signaling, but it is not clear whether this is species specific. Dogs with IVD disease could possibly benefit from Link-N treatment, but Link-N has not been tested on canine IVD cells. If Link-N appears to be effective in canines, this would facilitate translation of Link-N into the clinic using the dog as an in vivo large animal model for human IVD degeneration. Materials and methods: This study’s objective was to determine the effect of the human and canine variant of Link-N and short (s) Link-N on canine chondrocyte-like cells (CLCs) and compare this to those on already studied species, i.e. human and bovine CLCs. Extracellular matrix (ECM) production was determined by measuring glycosaminoglycan (GAG) content and histological evaluation. Additionally, the micro-aggregates’ DNA content was measured. Phosphorylated (p) Smad1 and -2 levels were determined using ELISA. Results: Human (s)Link-N induced GAG deposition in human and bovine CLCs, as expected. In contrast, canine (s)Link-N did not affect ECM production in human CLCs, while it mainly induced collagen type I and II deposition in bovine CLCs. In canine CLCs, both canine and human (s)Link-N induced negligible GAG deposition. Surprisingly, human and canine (s)Link-N did not induce Smad signaling in human and bovine CLCs. Human and canine (s)Link-N only mildly increased pSmad1 and Smad2 levels in canine CLCs. Conclusions: Human and canine (s)Link-N exerted species-specific effects on CLCs from early degenerated IVDs. Both variants, however, lacked the potency as canine IVD regeneration agent. While these studies demonstrate the challenges of translational studies in large animal models, (s)Link-N still holds a regenerative potential for humans. [ABSTRACT FROM AUTHOR]

Vasculoprotective endothelium glycocalyx (GCX) shedding plays a critical role in vascular disease. Previous work demonstrated that GCX degradation disrupts endothelial cell (EC) gap junction connexin (Cx) proteins, likely blocking interendothelial molecular transport that maintains EC and vascular tissue homeostasis to resist disease. Here, we focused on GCX regeneration and tested the hypothesis that vasculoprotective EC function can be stimulated via replacement of GCX when it is shed. We used EC with [i] intact heparan sulfate (HS), the most abundant GCX component; [ii] degraded HS; or [iii] HS that was restored after enzyme degradation, by cellular self-recovery or artificially. Artificial HS restoration was achieved via treatment with exogenous HS, with or without the GCX regenerator and protector sphingosine 1- phosphate (S1P). In these cells we immunocytochemically examined expression of Cx isotype 43 (Cx43) at EC borders and characterized Cx-containing gap junction activity by measuring interendothelial spread of gap junction permeable Lucifer Yellow dye. With intact HS, 60% of EC borders expressed Cx43 and dye spread to 2.88 ± 0.09 neighboring cells. HS degradation decreased Cx43 expression to 30% and reduced dye spread to 1.87± 0.06 cells. Cellular self-recovery of HS restored baseline levels of Cx43 and dye transfer. Artificial HS recovery with exogenous HS partially restored Cx43 expression to 46% and yielded dye spread to only 1.03 ± 0.07 cells. Treatment with both HS and S1P, recovered HS and restored Cx43 to 56% with significant dye transfer to 3.96 ± 0.23 cells. This is the first evidence of GCX regeneration in a manner that effectively restores vasculoprotective EC communication. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a progressive degenerative disease that results in fibrosis and atrophy of muscles. The main cause of death associated with DMD is failure of the diaphragm. The diaphragm is a dome-shaped muscle with a fiber microstructure that differs across regions of the muscle. However, no studies to our knowledge have examined spatial variations of muscle fibers in dystrophic diaphragm or how aging affects those variations in DMD. In this study, diaphragms were obtained from mdx and healthy mice at ages three, seven, and ten months in the dorsal, midcostal, and ventral regions. Through immunostaining and confocal imaging, we quantified sarcomere length, interstitial space between fibers, fiber branching, fiber cross sectional area (CSA), and fiber regeneration measured by centrally located nuclei. Because DMD is associated with chronic inflammation, we also investigated the number of macrophages in diaphragm muscle cross-sections. We saw regional differences in the number of regenerating fibers and macrophages during the progression of DMD in the mdx diaphragm. Additionally, the number of regenerating fibers increased with age, while CSA and the number of branching fibers decreased. Dystrophic diaphragms had shorter sarcomere lengths than age-matched controls. Our results suggest that the dystrophic diaphragm in the mdx mouse is structurally heterogeneous and remodels non-uniformly over time. Understanding regional changes in dystrophic diaphragms over time will facilitate the development of targeted therapies to prevent or minimize respiratory failure in DMD patients. [ABSTRACT FROM AUTHOR]

This study was designed to assess the effects of cold atmospheric plasma on osteoblastic differentiation in pre-osteoblastic MC3T3-E1 cells. Plasma was irradiated directly to a culture medium containing plated cells for 5 s or 10 s. Alkaline phosphatase (ALP) activity assay and alizarin red staining were applied to assess osteoblastic differentiation. The plasma-generated radicals were detected directly using an electron spin resonance-spin trapping technique. Results show that plasma irradiation under specific conditions increased ALP activity and enhanced mineralization, and demonstrated that the yield of radicals was increased in an irradiation-time-dependent manner. Appropriate plasma irradiation stimulated the osteoblastic differentiation of the cells. This process offers the potential of promoting bone regeneration. [ABSTRACT FROM AUTHOR]

Organ regeneration is becoming a promising choice for many patients; however, many details about the mechanisms underlying organ regeneration remain unknown. As regenerative organs, hair follicles offer a good model to study the mechanisms associated with regenerative medicine. The relevant studies have mainly focused on donor cells, and there are no systematic studies involving the effect of host factors on hair follicle reconstruction. Thus, we intend to explore the effect of host cells on hair follicle reconstruction. Epidermal and dermal cells from red fluorescent protein (RFP) transgenic newborn mice were injected into green fluorescent protein (GFP) transgenic mice. In addition, we wrapped the mixed dermal and epidermal cells from GFP transgenic and RFP transgenic mice by the Cell-in-a-Box kit to form "capsules," so that the cells within would be isolated from host cells. These capsules were cultured in vitro and transplanted in vivo. Fully developed reconstructed hair follicles were observed after the injection of mixed cells. These reconstructed follicles mainly consisted of donor cells, as well as a small number of host cells. The encapsulated cells gradually aggregated into cell spheres in vitro without apparent differentiation towards hair follicles. With respect to the transplanted capsules, concentric circle structures were observed, but no hair follicles or hair shafts formed. When the concentric circle structures were transplanted in vivo, mature hair follicles were observed 30 days later. Host cells were found in the reconstructed hair follicles. Thus, we conclude that host cells participate in the process of hair follicle reconstruction, and they play a vital role in the process, especially for the maturation of reconstructed hair follicles. Furthermore, we established a special hair follicle reconstruction system with the help of capsules: transplant cells were isolated from host, but other factors from host could exchange with cells inside. [ABSTRACT FROM AUTHOR]

Demyelination is observed in numerous diseases of the central nervous system, including multiple sclerosis (MS). However, the endogenous regenerative process of remyelination can replace myelin lost in disease, and in various animal models. Unfortunately, the process of remyelination often fails, particularly with ageing. Even when remyelination occurs, it is characterised by the regeneration of myelin sheaths that are abnormally thin and short. This imperfect remyelination is likely to have implications for the restoration of normal circuit function and possibly the optimal metabolic support of axons. Here we describe a larval zebrafish model of demyelination and remyelination. We employ a drug-inducible cell ablation system with which we can consistently ablate 2/3rds of oligodendrocytes in the larval zebrafish spinal cord. This leads to a concomitant demyelination of 2/3rds of axons in the spinal cord, and an innate immune response over the same time period. We find restoration of the normal number of oligodendrocytes and robust remyelination approximately two weeks after induction of cell ablation, whereby myelinated axon number is restored to control levels. Remarkably, we find that myelin sheaths of normal length and thickness are regenerated during this time. Interestingly, we find that axons grow significantly in caliber during this period of remyelination. This suggests the possibility that the active growth of axons may stimulate the regeneration of myelin sheaths of normal dimensions. [ABSTRACT FROM AUTHOR]

Background and aims: Antithrombin III (ATIII) has been reported to be associated with liver pathologies and was shown to predict outcome in patients undergoing liver resection for hepatocellular carcinoma. We now aimed to assess whether perioperative ATIII-activity could predict postoperative outcome in patients without underlying liver disease, as well as in a routine clinical setting of patients undergoing hepatic resection. Methods: ATIII-activity was evaluated preoperatively and on the first (POD1) and fifth day after liver resection in a retrospective evaluation cohort of 228 colorectal cancer patients with liver metastasis (mCRC). We further aimed to prospectively validate our results in a set of 177 consecutive patients undergoing hepatic resection. Results: Patients developing postoperative liver dysfunction (LD) had a more pronounced postoperative decrease in ATIII-activity (P<0.001). ATIII-activity on POD1 significantly predicted postoperative LD (P<0.001, AUC = 84.4%) and remained independent upon multivariable analysis. A cut-off value of 61.5% ATIII-activity was determined using ROC analysis. This cut-off was vital to identify high-risk patients for postoperative LD, morbidity, severe morbidity and mortality (P<0.001, respectively) with a highly accurate negative predictive value of 97%, which could be confirmed for LD (P<0.001) and mortality (P = 0.014) in our independent validation cohort. Further, mCRC patients below our cut-off suffered from a significantly decreased overall survival (OS) at 1 and 3 years after surgery (P = 0.011, P = 0.025). Conclusions: The routine laboratory parameter ATIII-activity on POD1 independently predicted postoperative LD and was associated with clinical outcome. Patients with a postoperative ATIII-activity <61.5% might benefit from close monitoring and timely initiation of supportive therapy. Trial registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Background: The primary therapeutic goals in the treatment of liver injury are to support liver regeneration or bridge the gap to liver transplantation (LT). Molecular adsorbent recirculating system (MARS) therapy has shown beneficial effects for specific symptoms of liver failure; however, general survival advantages have not yet been demonstrated. Aim: We studied the effects of MARS therapy compared to standard medical treatment (SMT) in two patient cohorts: in patients with an acute liver injury and in those with graft dysfunction (GD). Methods: We report on our experience over a 6.5-year period with 73 patients treated with SMT or with SMT and MARS (MARS group). In total, 53 patients suffered from acute liver injury in their native liver without a preexisting liver disease (SMT: n = 31, MARS: n = 22), and 20 patients showed a severe GD after LT (SMT: n = 10, MARS: n = 10). Results: The entire cohort was predominantly characterized by hemodynamically and respiratorily stable patients with a low hepatic encephalopathy (HE) grade and a model of end-stage liver disease (MELD) score of 20.57 (MARS) or 22.51 (SMT, p = 0.555). Within the MARS group, the median number of extracorporeal therapy sessions was four (range = 3–5 sessions). Independent of the underlying etiology, MARS improved the patients’ bilirubin values in the short term compared to SMT alone. In patients with acute liver injury, this response was sustained even after the end of MARS therapy. By contrast, the majority of patients with GD and an initial response to MARS therapy experienced worsened hyperbilirubinemia. No differences in 28-day mortality were observed with respect to acute liver injury (MARS 5.3% (95% CI: 0–15.3); SMT 3.3% (95% CI: 0–9.8), p = 0.754) or GD (MARS 20.0% (95% CI: 0–44.7), SMT 11.1% (95% CI: 0–31.7), p = 0.478). Conclusions: Although it did not improve 28-day mortality, MARS therapy improved the short-term response in patients with acute liver injury as well as in those with GD. In cases of acute hepatic injury, the use of MARS therapy resulted in the sustained stabilization of liver function and improved liver regeneration. A short-term response to MARS may predict the future course of the disease. [ABSTRACT FROM AUTHOR]

The multi-cellular nature of renal tissue makes it the most challenging organ for regeneration. Therefore, till date whole organ transplantations remain the definitive treatment for the end stage renal disease (ESRD). The shortage of available organs for the transplantation has, thus, remained a major concern as well as an unsolved problem. In this regard generation of whole organ scaffold through decellularization followed by regeneration of the whole organ by recellularization is being viewed as a potential alternative for generating functional tissues. Despite its growing interest, the optimal processing to achieve functional organ still remains unsolved. The biggest challenge remains is the time line for obtaining kidney. Keeping these facts in mind, we have assessed the effects of cryostorage (3 months) on renal tissue architecture and its potential for decellularization and recellularization in comparison to the freshly isolated kidneys. The light microscopy exploiting different microscopic stains as well as immuno-histochemistry and Scanning electron microscopy (SEM) demonstrated that ECM framework is well retained following kidney cryopreservation. The strength of these structures was reinforced by calculating mechanical stress which confirmed the similarity between the freshly isolated and cryopreserved tissue. The recellularization of these bio-scaffolds, with mesenchymal stem cells quickly repopulated the decellularized structures irrespective of the kidneys status, i.e. freshly isolated or the cryopreserved. The growth pattern employing mesenchymal stem cells demonstrated their equivalent recellularization potential. Based on these observations, it may be concluded that cryopreserved kidneys can be exploited as scaffolds for future development of functional organ. [ABSTRACT FROM AUTHOR]

Many recent studies have been conducted to assess the ability of composite materials containing carbon nanotubes (CNTs) with high bone affinity to serve as scaffolds in bone regenerative medicine. These studies have demonstrated that CNTs can effectively induce bone formation. However, no studies have investigated the usefulness of scaffolds consisting exclusively of CNTs in bone regenerative medicine. We built a three-dimensional block entity with maximized mechanical strength from multi-walled CNTs (MWCNT blocks) and evaluated their efficacy as scaffold material for bone repair. When MWCNT blocks containing recombinant human bone morphogenetic protein-2 (rhBMP-2) were implanted in mouse muscle, ectopic bone was formed in direct contact with the blocks. Their bone marrow densities were comparable to those of PET-reinforced collagen sheets with rhBMP-2. On day 1 and day 3, MC3T3-E1 preosteoblasts were attached to the scaffold surface of MWCNT blocks than that of PET-reinforced collagen sheets. They also showed a maximum compression strength comparable to that of cortical bone. Our MWCNT blocks are expected to serve as bone defect filler and scaffold material for bone regeneration. [ABSTRACT FROM AUTHOR]

The lung alveoli slowly self-renew pneumocytes, but their facultative regeneration capacity is rapidly efficient after an injury, so fibrosis infrequently occurs. We recently observed Keratin 14 (KRT14) expression during diffuse alveolar damage (DAD), but not in controls. We wonder if KRT14 may be a marker of pneumocyte transition from quiescence to regeneration. Quantitative PCR and Western blot analyses highlighted the presence of KRT14 (mRNA and protein) only in human lung samples with DAD or interstitial lung disease (ILD). In the exponentially growing cell lines A549 and H441, the mRNA and protein levels of KRT14 peaked at day one after cell seeding and decreased at day two, opposite to what observed for the proliferation marker E2F1. The inverse relation of KRT14 versus E2F1 expression holds true also for other proliferative markers, such as cyclin E1 and cyclin D1. Of interest, we also found that E2F1 silencing caused cell cycle arrest and increased KRT14 expression, whilst E2F1 stimulation induced cell cycle progression and decreased KRT14. KRT14 also increased in proliferative pneumocytes (HPAEpiC) just before transdifferentiation. Overall, our results suggest that KRT14 is a viable biomarker of pneumocyte activation, and repair/regeneration. The involvement of KRT14 in regenerative process may suggest a novel pharmaceutical target to accelerate lung repair. [ABSTRACT FROM AUTHOR]

Background: The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development. Methods: We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium. Results: The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described. Conclusions: Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas. [ABSTRACT FROM AUTHOR]

Development and maintenance of secondary lymphoid organs such as lymph nodes and spleen essentially depend on lymphotoxin β-receptor (LTβR) signaling. It is unclear, however, by which molecular mechanism their size is limited. Here, we investigate whether the LTβR pathway is also growth suppressing. By using splenic tissue transplantation it is possible to analyze a potential contribution of LTβR signaling inside and outside of the implanted tissue. We show that LTβR signaling within the endogenous spleen and within non-splenic tissues both significantly suppressed the regeneration of implanted splenic tissue. The suppressive activity positively correlated with the total number of LTβR expressing cells in the animal (regenerate weights of 115 ± 8 mg in LTβR deficient recipients and of 12 ± 9 mg in wild-type recipients), affected also developed splenic tissue, and was induced but not executed via LTβR signaling. Two-dimensional differential gel electrophoresis and subsequent mass spectrometry of stromal splenic tissue was applied to screen for potential factors mediating the LTβR dependent suppressive activity. Thus, LTβR dependent growth suppression is involved in regulating the size of secondary lymphoid organs, and might be therapeutically used to eradicate tertiary lymphoid tissues during autoimmune diseases. [ABSTRACT FROM AUTHOR]

The creation of engineered 3D microtissues has attracted prodigious interest because of the fact that this microtissue structure is able to mimic in vivo environments. Such microtissues can be applied extensively in the fields of regenerative medicine and tissue engineering, as well as in drug and toxicity screening. Here, we develop a novel method of fabricating a large number of dense honeycomb concave microwells via surface tension-mediated self-construction. More specifically, in order to control the curvature and shape of the concavity in a precise and reproducible manner, a custom-made jig system was designed and fabricated. By applying a pre-set force using the jig system, the shape of the honeycomb concave well was precisely and uniformly controlled, despite the fact that wells were densely packed. The thin wall between the honeycomb wells enables the minimization of cell loss during the cell-seeding process. To evaluate the performance of the honeycomb microwell array, rat hepatocytes were seeded, and spheroids were successfully formed with uniform shape and size. Liver-specific functions such as albumin secretion and cytochrome P450 were subsequently analyzed. The proposed method of fabricating honeycomb concave wells is cost-effective, simple, and reproducible. The honeycomb well array can produce multiple spheroids with minimal cell loss, and can lead to significant contributions in tissue engineering and organ regeneration. [ABSTRACT FROM AUTHOR]

The corneal endothelium maintains corneal transparency by its pump and barrier functions; consequently, its decompensation due to any pathological reason causes severe vision loss due to corneal haziness. Corneal transplantation is the only therapeutic choice for treating corneal endothelial dysfunction, but associated problems, such as a shortages of donor corneas, the difficulty of the surgical procedure, and graft failure, still need to be resolved. Regenerative medicine is attractive to researchers as a means of providing innovative therapies for corneal endothelial dysfunction, as it now does for other diseases. We previously demonstrated the successful regeneration of corneal endothelium in animal models by injecting cultured corneal endothelial cells (CECs) in combination with a Rho kinase (ROCK) inhibitor. The purpose of the present study was to optimize the vehicle for clinical use in cell-based therapy. Our screening of cell culture media revealed that RELAR medium promoted CEC adhesion. We then modified RELAR medium by removing hormones, growth factors, and potentially toxic materials to generate a cell therapy vehicle (CTV) composed of amino acid, salts, glucose, and vitamins. Injection of CECs in CTV enabled efficient engraftment and regeneration of the corneal endothelium in the rabbit corneal endothelial dysfunction model, with restoration of a transparent cornea. The CECs retained >85% viability after a 24 hour preservation as a cell suspension in CTV at 4°C and maintained their potency to regenerate the corneal endothelium in vivo. The vehicle developed here is clinically applicable for cell-based therapy aimed at treating the corneal endothelium. Our strategy involves the generation of vehicle from a culture medium appropriate for a given cell type by removing materials that are not favorable for clinical use. [ABSTRACT FROM AUTHOR]

Emerging evidences have revealed that long non-coding RNAs (lncRNAs) functioned in a wide range of physiological and pathophysiological processes including rat liver regeneration, and could regulate gene expression in the transcriptional and post-transcriptional levels. However, the underlying mechanism for lncRNAs participation in liver regeneration is largely unknown. To define the mechanisms how the lncRNAs regulate LR, we performed bio-chip technology, high-throughput sequencing and RT-PCR to detect the expression of lncRNAs at 0, 2 and 6 h during LR after 2/3 hepatectomy (PH). The results indicated that 28 lncRNAs were involved in LR. Bioinformatics analysis predicated 465 co-expression target genes including 10 regulatory genes were related to these 28 lncRNAs. Ingenuity Pathway Analysis (IPA) was employed to analyze the signaling pathways and physiological activities that regulated by these genes, and the results suggested that these genes were potentially related to ILK, SAPK/JNK and ERK/MAPK signaling pathways, and possibly regulate many important physiological activities in LR in terms of cell proliferation, cell differentiation, cell survival, apoptosis and necrosis. [ABSTRACT FROM AUTHOR]

Introduction: Osteoarthritis (OA) is the most common musculoskeletal disease. Current treatments for OA are mainly symptomatic and inadequate since none results in restoration of fully functional cartilage. Hyaluronic Acid (HA) intra-articular injections are widely accepted for the treatment of pain associated to OA. The goal of HA viscosupplementation is to reduce pain and improve viscoelasticity of synovial fluid. Platelet-rich plasma (PRP) has been also employed to treat OA to possibly induce cartilage regeneration. The combination of HA and PRP could supply many advantages for tissue repair. Indeed, it conjugates HA viscosupplementation with PRP regenerative properties. The aim of this study was to evaluate the rheological and biological properties of different HA compositions in combination with PRP in order to identify (i) the viscoelastic features of the HA-PRP blends, (ii) their biological effect on osteoarthritic chondrocytes and (iii) HA formulations suitable for use in combination with PRP. Materials and Methods: HA/PRP blends have been obtained mixing human PRP and three different HA at different concentrations: 1) Sinovial, 0.8% (SN); 2) Sinovial Forte 1.6% (SF); 3) Sinovial HL 3.2% (HL); 4) Hyalubrix 1.5% (HX). Combinations of phosphate buffered saline (PBS) and the four HA types were used as control. Rheological measurements were performed on an Anton PaarMCR-302 rheometer. Amplitude sweep, frequency sweep and rotational measurements were performed and viscoelastic properties were evaluated. The rheological data were validated performing the tests in presence of Bovine Serum Albumin (BSA) up to ultra-physiological concentration (7%). Primary osteoarthritic chondrocytes were cultured in vitro with the HA and PRP blends in the culture medium for one week. Cell viability, proliferation and glycosaminoglycan (GAG) content were assessed. Results: PRP addition to HA leads to a decrease of viscoelastic shear moduli and increase of the crossover point, due to a pure dilution effect. For viscosupplements with HA concentration below 1% the viscoelasticity is mostly lost. Results were validated also in presence of proteins, which in synovial fluid are more abundant than HA. Chondrocytes proliferated overtime in all different culture conditions. The proliferation rate was higher in chondrocytes cultured in the media containing PRP compared to the cultures with different HA alone. GAG content was significantly higher in chondrocytes cultured in PRP and HL blend. Discussion: We investigated the rheological and biological properties of four different HA concentrations when combined with PRP giving insights on viscoelastic and biological properties of a promising approach for future OA therapy. Our data demonstrate that PRP addition is not detrimental to the viscosupplementation effect of HA. Viscosupplements containing low HA concentration are not indicated for combination with PRP, as the viscoelastic properties are lost. Although having the same rheological behavior of SF and HX, HL was superior in stimulating extracellular matrix production in vitro. [ABSTRACT FROM AUTHOR]

Regeneration of periodontal tissues requires a concerted effort to obtain consistent and predictable results in vivo. The aim of the present study was to test a new family of bioactive polymeric membranes in combination with stem cell therapy for periodontal regeneration. In particular, the novel polyester poly(isosorbide succinate-co-L-lactide) (PisPLLA) was compared with poly(L-lactide) (PLLA). Both polymers were combined with collagen (COL), hydroxyapatite (HA) and the growth factor bone morphogenetic protein-7 (BMP7), and their osteoinductive capacity was evaluated via in vitro and in vivo experiments. Membranes composed of PLLA/COL/HA or PisPLLA/COL/HA were able to promote periodontal regeneration and new bone formation in fenestration defects in rat jaws. According to quantitative real-time polymerase chain reaction (qRT-PCR) and Alizarin Red assays, better osteoconductive capacity and increased extracellular mineralization were observed for PLLA/COL/HA, whereas better osteoinductive properties were associated with PisPLLA/COL/HA. We concluded that membranes composed of either PisPLLA/COL/HA or PLLA/COL/HA present promising results in vitro as well as in vivo and that these materials could be potentially applied in periodontal regeneration. [ABSTRACT FROM AUTHOR]

Complete reconstruction of damaged periodontal pockets, particularly regeneration of periodontal ligament (PDL) has been a significant challenge in dentistry. Tissue engineering approach utilizing PDL stem cells and scaffolding matrices offers great opportunity to this, and applying physical and mechanical cues mimicking native tissue conditions are of special importance. Here we approach to regenerate periodontal tissues by engineering PDL cells supported on a nanofibrous scaffold under a mechanical-stressed condition. PDL stem cells isolated from rats were seeded on an electrospun polycaprolactone/gelatin directionally-oriented nanofiber membrane and dynamic mechanical stress was applied to the cell/nanofiber construct, providing nanotopological and mechanical combined cues. Cells recognized the nanofiber orientation, aligning in parallel, and the mechanical stress increased the cell alignment. Importantly, the cells cultured on the oriented nanofiber combined with the mechanical stress produced significantly stimulated PDL specific markers, including periostin and tenascin with simultaneous down-regulation of osteogenesis, demonstrating the roles of topological and mechanical cues in altering phenotypic change in PDL cells. Tissue compatibility of the tissue-engineered constructs was confirmed in rat subcutaneous sites. Furthermore, in vivo regeneration of PDL and alveolar bone tissues was examined under the rat premaxillary periodontal defect models. The cell/nanofiber constructs engineered under mechanical stress showed sound integration into tissue defects and the regenerated bone volume and area were significantly improved. This study provides an effective tissue engineering approach for periodontal regeneration—culturing PDL stem cells with combinatory cues of oriented nanotopology and dynamic mechanical stretch. [ABSTRACT FROM AUTHOR]

Electroactive materials have been investigated as next-generation neuronal tissue engineering scaffolds to enhance neuronal regeneration and functional recovery after brain injury. Graphene, an emerging neuronal scaffold material with charge transfer properties, has shown promising results for neuronal cell survival and differentiation in vitro. In this in vivo work, electrospun microfiber scaffolds coated with self-assembled colloidal graphene, were implanted into the striatum or into the subventricular zone of adult rats. Microglia and astrocyte activation levels were suppressed with graphene functionalization. In addition, self-assembled graphene implants prevented glial scarring in the brain 7 weeks following implantation. Astrocyte guidance within the scaffold and redirection of neuroblasts from the subventricular zone along the implants was also demonstrated. These findings provide new functional evidence for the potential use of graphene scaffolds as a therapeutic platform to support central nervous system regeneration. [ABSTRACT FROM AUTHOR]

The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process. [ABSTRACT FROM AUTHOR]

Background: A longstanding goal in regenerative medicine is to reconstitute functional tissus or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised. Methods: We have used transgenic Tg:Pax7nGFP and Flk1GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex®. Results: We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a “dead zone” devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models. Conclusions: Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Furthermore, we show that histological parameters are not wholly sufficient to declare that regeneration is complete as molecular alterations (e.g. cycling SCs, cytokines) could have a major persistent impact. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to examine the influence of particle size and extent of demineralization of dentin matrix on bone regeneration. Materials and Methods: Extracted human teeth were pulverized and divided into 3 groups according to particle size; 200, 500, and 1000 μm. Each group was divided into 3 groups depending on the extent of demineralization; undemineralized dentin (UDD), partially demineralized dentin matrix (PDDM), and completely demineralized dentin matrix (CDDM). The dentin sample was implanted into rat calvarial bone defects. After 4 and 8 weeks, the bone regeneration was evaluated with micro-CT images, histomorphometric and immunohistochemical analyses. Osteoblasts were cultured on UDD and DDM to evaluate the cell attachment using electron microscope. Results: Micro-CT images and histological observation revealed that CDDM had largely resorbed but UDD had not, and both of them induced little bone formation, whereas all particle sizes of PDDM induced more new bone, especially the 1000 μm. Electron microscopic observation showed osteoblasts attached to DDM but not to UDD. Conclusions: PDDM with larger particle size induced prominent bone regeneration, probably because PDDM possessed a suitable surface for cell attachment. There might be an exquisite balance between its resorption and bone formation on it. PDDM could be considered as a potential bone substitute. [ABSTRACT FROM AUTHOR]

The fruit fly, Drosophila melanogaster, is a commonly used model organism for neurodegenerative diseases. Its major advantages include a short lifespan and its susceptibility to manipulation using sophisticated genetic techniques. Here, we report the systematic comparison of fly models of two polyglutamine (polyQ) diseases. We induced expression of the normal and mutant forms of full-length Ataxin-1 and Huntingtin exon 1 in cholinergic, dopaminergic, and motor neurons, and glial cells using cell type-specific drivers. We systematically analyzed their effects based on multiple phenotypes: eclosion rate, lifespan, motor performance, and circadian rhythms of spontaneous activity. This systematic assay system enabled us to quantitatively evaluate and compare the functional disabilities of different genotypes. The results suggest different effects of Ataxin-1 and Huntingtin on specific types of neural cells during development and in adulthood. In addition, we confirmed the therapeutic effects of LiCl and butyrate using representative models. These results support the usefulness of this assay system for screening candidate chemical compounds that modify the pathologies of polyQ diseases. [ABSTRACT FROM AUTHOR]