Britani N. Blackstone

Plasma surface modification of electrospun fibers for adhesion-based cancer cell sorting

Personalized cancer therapies drive the need for devices that rapidly and accurately segregate cancer cells from solid tumors. One potential sorting strategy is to segregate populations of cells based on their relative strength of adhesion. To investigate the effect of surface hydrophilicity and cell phenotype on adhesion, primary human breast skin fibroblasts and keratinocytes and MCF-7 breast cancer cells were seeded onto air and CF(4) plasma-treated nanofibers followed by exposure to three shear stresses (200, 275 and 350 dynes per cm(2)) 1 hour after inoculation. No difference in strength of adhesion was measured in either fibroblasts or keratinocytes on either plasma treated-surface: all exhibited >60% of the initial cell count after a 5 minute exposure to 350 dynes per cm(2) of shear stress. In contrast, a significant difference between relative strength of adhesion on air versus CF(4) plasma-treated surfaces was observed for MCF-7 cells: 26% and 6.6% of cells remained on the air and CF(4) plasma-treated surfaces, respectively. The ability to sort this cancer cell line from two non-cancerous primary human cells was evaluated by inoculating a mixture of all three cell types simultaneously onto CF(4) treated nanofibers followed by 1 hour of culture and exposure to 350 dynes per cm(2) shear stress. The majority of MCF-7 cells were removed (0.7% remained) while a majority of fibroblasts and keratinocytes remained adhered (74 and 57%). Post-sorted MCF-7 viability and morphology remained unchanged, preserving the possibility of post-separation and analysis. These data suggest that the plasma treatment of electrospun scaffolds provides a tool useful in sorting cancer cells from a mixed cell population based on adhesion strength.

Inflammatory responses, matrix remodeling, and re‐epithelialization after fractional CO2 laser treatment of scars

Fractional CO2 laser therapy has been used to improve scar pliability and appearance; however, a variety of treatment protocols have been utilized with varied outcomes. Understanding the relationship between laser power and extent of initial tissue ablation and time frame for remodeling could help determine an optimum power and frequency for laser treatment. The characteristics of initial injury caused by fractional CO2 laser treatment, the rates of dermal remodeling and re‐epithelialization, and the extent of inflammation as a function of laser stacking were assessed in this study in a porcine scar model.

Effects of early combinatorial treatment of autologous split‐thickness skin grafts in red duroc pig model using pulsed dye laser and fractional CO2 laser

The use of pulsed dye laser (PDL) and fractional CO2 (FX CO2) laser therapy to treat and/or prevent scarring following burn injury is becoming more widespread with a number of studies reporting reduction in scar erythema and pruritus following treatment with lasers. While the majority of studies report positive outcomes following PDL or FX CO2 therapy, a number of studies have reported no benefit or worsening of the scar following treatment. The objective of this study was to directly compare the efficacy of PDL, FX CO2, and PDL + FX CO2 laser therapy in reducing scarring post burn injury and autografting in a standardized animal model.

Myoferlin depletion elevates focal adhesion kinase and paxillin phosphorylation and enhances cell-matrix adhesion in breast cancer cells

Breast cancer is the second leading cause of malignant death among women. A crucial feature of metastatic cancers is their propensity to lose adhesion to the underlying basement membrane as they transition to a motile phenotype and invade surrounding tissue. Attachment to the extracellular matrix is mediated by a complex of adhesion proteins, including integrins, signaling molecules, actin and actin-binding proteins, and scaffolding proteins. Focal adhesion kinase (FAK) is pivotal for the organization of focal contacts and maturation into focal adhesions, and disruption of this process is a hallmark of early cancer invasive potential. Our recent work has revealed that myoferlin (MYOF) mediates breast tumor cell motility and invasive phenotype. In this study we demonstrate that noninvasive breast cancer cell lines exhibit increased cell-substrate adhesion and that silencing of MYOF using RNAi in the highly invasive human breast cancer cell line MDA-MB-231 also enhances cell-substrate adhesion. In addition, we detected elevated tyrosine phosphorylation of FAK (FAK(Y397)) and paxillin (PAX(Y118)), markers of focal adhesion protein activation. Morphometric analysis of PAX expression revealed that RNAi-mediated depletion of MYOF resulted in larger, more elongated focal adhesions, in contrast to cells transduced with a control virus (MDA-231(LVC) cells), which exhibited smaller focal contacts. Finally, MYOF silencing in MDA-MB-231 cells exhibited a more elaborate ventral cytoskeletal structure near focal adhesions, typified by pronounced actin stress fibers. These data support the hypothesis that MYOF regulates cell adhesions and cell-substrate adhesion strength and may account for the high degree of motility in invasive breast cancer cells.

Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue.

Inflammation, following injury, induces cellular plasticity as an inherent component of physiological tissue repair. The dominant fate of wound macrophages is unclear and debated. Here we show that two-thirds of all granulation tissue fibroblasts, otherwise known to be of mesenchymal origin, are derived from myeloid cells which are likely to be wound macrophages. Conversion of myeloid to fibroblast-like cells is impaired in diabetic wounds. In cross-talk between keratinocytes and myeloid cells, miR-21 packaged in extracellular vesicles (EV) is required for cell conversion. EV from wound fluid of healing chronic wound patients is rich in miR-21 and causes cell conversion more effectively compared to that by fluid from non-healing patients. Impaired conversion in diabetic wound tissue is rescued by targeted nanoparticle-based delivery of miR-21 to macrophages. This work introduces a paradigm wherein myeloid cells are recognized as a major source of fibroblast-like cells in the granulation tissue.At the site of injury, macrophages exit their characteristic phenotype undergoing direct conversion to fibroblasts. Keratinocyte-derived miR-21, packaged in extracellular vesicles, enables such plasticity which accounts for the vast majority of all fibroblasts in the granulation tissue.

Structural Alterations in Scaffold Architecture in Response to Mechanical Stimuli

Background: Though mechanical stimulation has been shown to improve mechanical properties of many engineered tissues, little is known about the impact of stimulation on scaffold architecture. In engineered tissues where the scaffold comprises a large portion of the tissue, mechanical signals are transferred from the external environment through the scaffold to the cells. Thus, a greater understanding of the architectural changes a scaffold experiences during mechanical stimulation may provide new knowledge on the communication between the cells and scaffold, during dynamic in vitro tissue development. Methods: Two distinct scaffold architectures were fabricated via lyophilization or electro-spinning of collagen. Pore size of lyophilized scaffolds, fiber diameter and inter-fiber distance of electrospun scaffolds, and ultimate tensile strength, linear stiffness and stress relaxation rates for all scaffolds were determined, prior to mechanical stimulation. Scaffolds were then subjected to 0, 5, 10 or 20% static or cyclic strain. Alignment of lyophilized scaffold pores and electrospun fibers was then quantified after 4 and 7 days of mechanical stimulation. Results: Lyophilized scaffolds displayed significant increases in pore area with magnitude of strain and duration of stimulation, and a pronounced alignment in pore orientation with the direction of strain. In contrast, electrospun samples showed only modest changes in architecture, in response to applied mechanical strain with small (1-5%) increased in fiber alignment compared to control, and no observed changed in fiber morphology. Conclusion: The current study showed the initial degree of interconnectivity between scaffold elements greatly impacted the scaffold response to mechanical stimulation. Non-woven electro-spun scaffold undergo very little micro structural reorganization during mechanical stimulation, while lyophilized collagen scaffolds undergo significant micro-structural changes. These differences in scaffold response suggest that large stain magnitudes may be required to excite cells within electro spun scaffolds, while modest strain magnitudes may results in significant changes in cellular behavior within lyophilized collagen sponges.

Early cessation of pressure garment therapy results in scar contraction and thickening

Pressure garment therapy is often prescribed to improve scar properties following full-thickness burn injuries. Pressure garment therapy is generally recommended for long periods of time following injury (1–2 years), though it is plagued by extremely low patient compliance. The goal of this study was to examine the effects of early cessation of pressure garment therapy on scar properties. Full-thickness burn injuries were created along the dorsum of red Duroc pigs. The burn eschar was excised and wound sites autografted with split-thickness skin. Scars were treated with pressure garments within 1 week of injury and pressure was maintained for either 29 weeks (continuous pressure) or for 17 weeks followed by cessation of pressure for an additional 12 weeks (pressure released); scars receiving no treatment served as controls. Scars that underwent pressure garment therapy were significantly smoother and less contracted with decreased scar height compared to control scars at 17 weeks. These benefits were maintained in the continuous pressure group until week 29. In the pressure released group, grafts significantly contracted and became more raised, harder and rougher after the therapy was discontinued. Pressure cessation also resulted in large changes in collagen fiber orientation and increases in collagen fiber thickness. The results suggest that pressure garment therapy effectively improves scar properties following severe burn injury; however, early cessation of the therapy results in substantial loss of these improvements.

Structural, Chemical, and Mechanical Properties of Pressure Garments as a Function of Simulated Use and Repeated Laundering

Pressure garments are widely employed for management of postburn scarring. Although pressure magnitude has been linked to efficacy, maintenance of uniform pressure delivery is challenging. An understanding of garment fabric properties is needed to optimize pressure delivery for the duration of garment use. To address this issue, compression vests were manufactured using two commonly used fabrics, Powernet or Dri-Tek Tricot, to achieve 10% reduction in circumference for a child-sized mannequin. Applied pressure was tracked on five anatomical sites over 23 hours, before laundering or after one and five laundering cycles. Load relaxation and fatigue of fabrics were tested before laundering or after one and five laundering cycles, and structural analysis via scanning electron microscopy was performed. Prior to laundering, pressure vests fabricated using Powernet or Dri-Tek Tricot generated a maximum pressure on the mannequin of 20 and 23 mm Hg, respectively. With both fabrics, pressure decreased during daily wear. Following five laundering cycles, Dri-Tek Tricot vests delivered a maximum of 7 vs 15 mm Hg pressure for Powernet at the same site. In cyclic tensile and load relaxation tests, exerted force correlated with fabric weave orientation with greatest force measured parallel to a fabrics long axis. The results demonstrate that Powernet exhibited the greatest applied force with the least garment fatigue. Fabric orientation with respect to the primary direction of tension was a critical factor in pressure generation and maintenance. This study suggests that fabrication of garments using Powernet with its long axis parallel to patients body part circumference may enhance the magnitude and maintenance of pressure delivery.

Inflammatory response and biomechanical properties of coaxial scaffolds for engineered skin in vitro and post-grafting

Engineered skin (ES) offers many advantages over split-thickness skin autografts for the treatment of burn wounds. However, ES, both in vitro and after grafting, is often significantly weaker, less elastic and more compliant than normal human skin. Biomechanical properties of ES can be tuned in vitro using electrospun co-axial (CoA) scaffolds. To explore the potential for coaxial scaffold-based ES use in vivo, two CoA scaffolds were fabricated with bioactive gelatin shells and biodegradable synthetic cores of polylactic acid (PLA) and polycaprolactone (PCL), and compared with gelatin monofilament scaffolds. Fibroblast and macrophage production of inflammatory cytokines interleukin 6 (IL-6) and transforming growth factor β-1 was significantly higher when cultured on PLA and PCL monofilament scaffolds compared to gelatin monofilament scaffolds. The core-shell fiber configuration significantly reduced production of pro-inflammatory cytokines to levels similar to those of gelatin monofilament scaffolds. In vitro, ES mechanical properties were significantly enhanced using CoA scaffolds; however, after grafting CoA- and gelatin-based ES to full-thickness excisional wounds on athymic mice, the in vitro mechanical advantage of CoA grafts was lost. A substantially increased inflammatory response to CoA-based ES was observed, with upregulation of IL-6 expression and a significant M2 macrophage presence. Additionally, expression of matrix metalloproteinase I was upregulated and collagen type I alpha 1 was downregulated in CoA ES two weeks after grafting. These results suggest that while coaxial scaffolds provide the ability to regulate biomechanics in vitro, further investigation of the inflammatory response to core materials is required to optimize this strategy for clinical use. STATEMENT OF SIGNIFICANCE: Engineered skin has been used to treat very large burn injuries. Despite its ability to heal these wounds, engineered skin exhibits reduced biomechanical properties making it challenging to manufacture and surgically apply. Coaxial fiber scaffolds have been utilized to tune the mechanical properties of engineered skin while maintaining optimal biological properties but it is not known how these perform on a patient especially with regards to their inflammatory response. The current study examines the biomechanical and inflammatory properties of coaxial scaffolds and uniaxial scaffolds in vitro and in vivo. The results show that the biological response to the scaffold materials is a critical determinant of tissue properties after grafting with reduced inflammation and rapid scaffold remodeling leading to stronger skin.

Abstract 5180: RNAi-mediated diminution of myoferlin increases adhesion of breast cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Ferlins are a family of proteins that play a variety of roles associated with plasma membrane dynamics in eukaryotes, including plasma membrane repair, regulation of membrane expression of receptors, and endocytosis. Dysferlin was recently reported to regulate platelet endothelial cellular adhesion molecule-1 (PECAM-1) in endothelial cells (1). We have shown that ablation of myoferlin (MYOF) in breast tumor cells attenuates invasion in vitro (in review, J Cell Sci). Moreover, loss of MYOF was found to be associated with reduced phosphorylation of a number of receptor tyrosine kinases (in press, PNAS), and others have reported on the role of MYOF in tyrosine kinase receptors expression at the plasma membrane (2). These results prompted us to hypothesize that MYOF may be involved in cell-matrix adhesion. Methods & Results: We used lentiviral-mediated RNA interference (RNAi) gene silencing to generate stable MYOF-deficient MDA-MB-231 cells and confirmed MYOF reduction with immunoblotting and qRT-PCR. RNAi control cells were generated in tandem with a non-human gene targeting construct. Relative adhesion and spreading of the wild-type, control, and MYOF-deficient epithelial cells were evaluated using a real-time electrical impedance measurement system (xCELLigence, Roche Applied Science). Results show a significant increase in the adhesion and spreading of the MYOF-deficient cells in comparision to wild-type and control cells. A trypsin assay developed using the same electrical impedance system demonstrated greater adhesion strength in the MYOF-deficient cells. This was measured by the duration of time necessary to reduce the pre-trypsin-treatment impedance by one-half. These results were further validated using a conventional parallel plate device, in which MYOF-deficient cells were found to be more adherent than controls when subjected to a range of shear stresses (1-10 dynes/cm2). PCR array screening of extracellular matrix related genes (SABiosciences) in MYOF-deficient and RNAi control MDA-MB-231 cells indicated that intercellular adhesion molecule 1 (ICAM-1/CD54) and vitronectin mRNA levels are increased following MYOF-diminution, and may potentially contribute to the mechanism behind the altered adhesion in MYOF-deficient cells. Conclusion: Myoferlin is important for the regulation of breast cancer epithelial cell adhesion. Since dysregulation of adhesion is often associated with cancer invasion and metastasis, myoferlin may play a role in these processes in breast carcinoma. (1) Sharma A, Yu C, Leung C, Trane A, Lau M, Utokaparch S, et al. A new role for the muscle repair protein dysferlin in endothelial cell adhesion and angiogenesis. Arterioscler Thromb Vasc Biol 2010 Nov;30 (11):2196-204. (2) Yu C, Sharma A, Trane A, Utokaparch S, Leung C, Bernatchez P. Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo. Vascul Pharmacol 2011 May 6. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5180. doi:1538-7445.AM2012-5180