Spinal motor neurite outgrowth over glial scar inhibitors is enhanced by coculture with bone marrow stromal cells

Background context Transplantation of bone marrow cells into spinal cord lesions promotes functional recovery in animal models, and recent clinical trials suggest possible recovery also in humans. The mechanisms responsible for these improvements are still unclear. Purpose To characterize spinal cord motor neurite interactions with human bone marrow stromal cells (MSCs) in an in vitro model of spinal cord injury (SCI). Study design/setting Previously, we have reported that human MSCs promote the growth of extending sensory neurites from dorsal root ganglia (DRG), in the presence of some of the molecules present in the glial scar, which are attributed with inhibiting axonal regeneration after SCI. We have adapted and optimized this system replacing the DRG with a spinal cord culture to produce a central nervous system (CNS) model, which is more relevant to the SCI situation. Methods We have developed and characterized a novel spinal cord culture system. Human MSCs were cocultured with spinal motor neurites in substrate choice assays containing glial scar-associated inhibitors of nerve growth. In separate experiments, MSC-conditioned media were analyzed and added to spinal motor neurites in substrate choice assays. Results As has been reported previously with DRG, substrate-bound neurocan and Nogo-A repelled spinal neuronal adhesion and neurite outgrowth, but these inhibitory effects were abrogated in MSC/spinal cord cocultures. However, unlike DRG, spinal neuronal bodies and neurites showed no inhibition to substrates of myelin-associated glycoprotein. In addition, the MSC secretome contained numerous neurotrophic factors that stimulated spinal neurite outgrowth, but these were not sufficient stimuli to promote spinal neurite extension over inhibitory concentrations of neurocan or Nogo-A. Conclusions These findings provide novel insight into how MSC transplantation may promote regeneration and functional recovery in animal models of SCI and in the clinic, especially in the chronic situation in which glial scars (and associated neural inhibitors) are well established. In addition, we have confirmed that this CNS model predominantly comprises motor neurons via immunocytochemical characterization. We hope that this model may be used in future research to test various other potential interventions for spinal injury or disease states. © 2014 Elsevier Inc. All rights reserved.

Bioengineered Approaches to Prevent Hypertrophic Scar Contraction

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.

In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.

In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.

In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.

In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.

Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.

The Land of the Savior: Óscar Romero and the Reform of Agriculture

This study approaches Óscar Romero by attending to his intimate involvement in and concern for the problematic surrounding the reform of Salvadoran agriculture and the conflict over property and possession underlying it. In this study, I situate Romero in relation to the concentration of landholding and the production of landlessness in El Salvador over the course of the twentieth century, and I examine his participation in the longstanding societal and ecclesial debate about agrarian reform provoked by these realities. I try to show how close attention to agrarian reform and what was at stake in it can illumine not only the conflict that occasioned Romero’s martyrdom but the meaning of the martyrdom itself.

Understanding Romero’s involvement in the debate about agrarian reform requires sustained attention to how it takes its bearings from the line of thinking about property and possession for which Pope Leo XIII’s 1891 encyclical Rerum novarum stands as a new beginning. The enclyclical tradition developing out of Leo’s pontificate is commonly referred to as Catholic social doctrine or Catholic social teaching. Romero’s and the Church’s participation in the debate about agrarian reform in El Salvador is unintelligible apart from it.

What Romero and the encyclical tradition share, I argue, is an understanding of creation as a common gift, from which follows a distinctive construal of property and the demands of justice with respect to possessing it. On this view, property does not name, as it is often taken to mean, the enclosure of what is common for the exclusive use of its possessors—something to be held by them over and against others. Rather, property and everything related to its holding derive from the claim that creation is a gift given to human creatures in common. The acknowledgement of creation as a common gift gives rise to what I describe in this study as a politics of common use, of which agrarian reform is one expression.

In Romero’s El Salvador, those who took the truth of creation as common gift seriously—those who spoke out against or opposed the ubiquity of the concentration of land and who clamored for agrarian reform so that the landless and land-poor could have access to land to cultivate for subsistence—suffered greatly as a consequence. I argue that, among other things, their suffering shows how, under the conditions of sin and violence, those who work to ensure that others have access to what is theirs in justice often risk laying down their lives in charity. In other words, they witness to the way that God’s work to restore creation has a cruciform shape. Therefore, while the advocacy for agrarian reform begins with the understanding of creation as common gift, the testimony to this truth in word and in deed points to the telos of the gift and the common life in the crucified and risen Lord in which it participates

Oceanographic data, oxygen and methane concentrations, and C-CH4 isotope ratios measured at Jaco Scar

Methane (CH4) concentrations and CH4 stable carbon isotopic composition (d13CCH4) were investigated in the water column within Jaco Scar. It is one of several scars formed by massive slides resulting from the subduction of seamounts offshore Costa Rica, a process that can open up structural and stratigraphical pathways for migrating CH4. The release of large amounts of CH4 into the adjacent water column was discovered at the outcropping lowermost sedimentary sequence of the hanging wall in the northwest corner of Jaco Scar, where concentrations reached up to 1,500 nmol L-1. There CH4-rich fluids seeping from the sedimentary sequence stimulate both growth and activity of a dense chemosynthetic community. Additional point sources supplying CH4 at lower concentrations were identified in density layers above and below the main plume from light carbon isotope ratios. The injected CH4 is most likely a mixture of microbial and thermogenic CH4 as suggested by d13CCH4 values between -50 and -62 per mil Vienna Pee Dee Belemnite. This CH4 spreads along isopycnal surfaces throughout the whole area of the scar, and the concentrations decrease due to mixing with ocean water and microbial oxidation. The supply of CH4 appears to be persistent as repeatedly high CH4 concentrations were found within the scar over 6 years. The maximum CH4 concentration and average excess CH4 concentration at Jaco Scar indicate that CH4 seepage from scars might be as significant as seepage from other tectonic structures in the marine realm. Hence, taking into account the global abundance of scars, such structures might constitute a substantial, hitherto unconsidered contribution to natural CH4 sources at the seafloor.

Characterization of the glial scar tissue in a murine model of spinal cord compression, with focus on hyaluronan

Spinal cord injury (SCI) is a devastating neurological disorder that affects thousands of people each year. Although in recent decades significant progress has been made in relation to understanding the molecular and cellular events underlying the nervous damage, spinal cord injury is still a highly disabling condition for which there is no curative therapy. People affected by spinal cord injuries manifested dysfunction or loss, temporary or permanent, of motor, sensory and / or autonomic functions depending on the spinal lesion damaged. Currently, the incidence rate of this type of injury is approximately 15-40 cases per million people worldwide. At the origin of these lesions are: road accidents, falls, interpersonal violence and the practice of sports. In this work we placed the hypothesis that HA is one of the component of the scar tissue formed after a compressive SCI, that it is likely synthetised by the perilesional glial cells and that it might support the permeation of the glial scar during the late phase of SCI. Nowadays, much focus is drawn on the recovery of CNS function, made impossible after SCI due to the high content of sulfated proteoglycans in the extracellular matrix. Counterbalancing the ratio between these proteoglycans and hyaluronic acid could be one of the experimental therapy to re-permeate the glial scar tissue formed after SCI, making possible axonal regrowth and functional recovery. Therefore, we established a model of spinal cord compression in mice and studied the glial scar tissue, particularly through the characterization of the expression of enzymes related to the metabolism of HA and the subsequent concentration thereof at different distances of the lesion epicenter. Our results show that the lesion induced in mice shows results similar to those produced in human lesions, in terms of histologic similarities and behavioral results. but these animals demonstrate an impressive spontaneous reorganization mechanism of the spinal cord tissue that occurs after injury and allows for partial recovery of the functions of the CNS. As regards the study of the glial scar, changes were recorded at the level of mRNA expression of enzymes metabolizing HA i.e., after injury there was a decreased expression of HA synthases 1-2 (HAS 1-2) and an increase of the expression HAS3 synthase mRNA, as well as the enzymes responsible for the HA catabolism, HYAL 1-2. But the amount of HA measured through the ELISA test was found unchanged after injury, it is not possible to explain this fact only with the change of expression of enzymes. At two weeks and in response to SCI, we found synthesized HA by reactive astrocytes and probably by others like microglial cells as it was advanced by the HA/GFAP+ and HA/IBA1+ cells co-location.

Photoletter to the editor: Calcinosis cutis in a burn scar

Calcinosis cutis is a rare condition characterized by the deposition of insoluble calcium salts in the skin and subcutaneous tissue. Dystrophic calcinosis cutis appears as a result of local tissue damage or abnormalities, such as alterations in extra-cellular matrix proteins or subcutaneous tissue with normal calcium and phosphate serum levels. It has been rarely described as a late complication of burns. Latency periods of 15-54 years have been reported. We describe the case of a 57-year-old man with dystrophic calcinosis cutis in a burn scar, which developed 42 years after the skin injury. The condition was successfully treated with surgical excision.

Recombinant human endostatin reduces hypertrophic scar formation in rabbit ear model through down- regulation of VEGF and TIMP-1.

Background: Recombinant human endostatin (Endostar) has been widely used to suppress angiogenesis in carcinoma patients. Hypertrophic scar (HS) tissue, much like a carcinoma, is often associated with angiogenesis. However, there have been few studies conducted on the effects of Endostar on HS or its mechanism. Objective: This paper investigated the effects Endostar on the HS of rabbit ears and studied the effects of Endostar on VEGF and TIMP-1 expression. Methods: Sixteen New Zealand white rabbits were used to establish HS models. Then, rabbit ears containing HS were randomly assigned to either the Endostar group or the control group. The changes of appearance and histology were evaluated using the naked eye, hematoxylin eosin staining, and a scar elevation index. The VEGF and TIMP-1 expressions were detected by immunohistochemical staining, RT-PCR, and western blot. Results: The thickness of the connective tissue in the Endostar group were thinner, the numbers of micro vessels and fibroblasts were fewer, and the collagen fibers were smoother. Moreover, the mRNA and protein expressions of VEGF and TIMP-1 in the Endostar group were significantly lower than those in the control group. Conclusion: The results suggested that Endostar reduced the formation of HS by down-regulation of VEGF and TIMP-1 expressions.