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Vocal fold lamina propria extracellular matrix (ECM) is highly aligned and when injured, becomes disorganized with loss of the tissue's critical biomechanical properties. This study examines the effects of electrospun fiber scaffold architecture and elastin-like polypeptide (ELP4) coating on human vocal fold fibroblast (HVFF) behavior for applications toward tissue engineering the vocal fold lamina propria. Electrospun Tecoflex™ scaffolds were made with aligned and unaligned fibers, and were characterized using scanning electron microscopy and uniaxial tensile testing. ELP4 was successfully adsorbed onto the scaffolds; HVFFs were seeded and their viability, proliferation, morphology and gene expression were characterized. Aligned and unaligned scaffolds had initial elastic moduli of ∼14 MPa, ∼5 MPa and ∼0.3 MPa, ∼0.6 MPa in the preferred and cross-preferred directions, respectively. Scaffold topography had an effect on the orientation of the cells, with HVFFs seeded on aligned scaffolds having a significantly different (p<0.001) angle of orientation than HVFFs cultured on unaligned scaffolds. This same effect and significant difference (p<0.001) was seen on aligned and unaligned scaffolds coated with ELP4. Scaffold alignment and ELP4 coating impacted ECM gene expression. ELP4 coating, and aligned scaffolds upregulated elastin synthesis when tested on day 7 without a concomitant upregulation of collagen III synthesis. Collectively, results indicate that aligned electrospun scaffolds and ELP4 coating are promising candidates in the development of biodegradeable vocal fold lamina propria constructs., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A tissue-engineered substitute that facilitates large-volume regeneration of the subcutaneous adipose tissue layer is needed for reconstructive plastic surgery. Towards this goal, we describe the in vitro culture of primary human adipose-derived stem cells (ASC) seeded into placental decellular matrix (PDM) and cross-linked hyaluronan (XLHA) scaffolds. Specifically, we evaluated cellular proliferation and adipogenic differentiation in the PDM, XLHA, and PDM combined with XLHA scaffolds. Cellular proliferation, viability, and glucose consumption were determined prior to the induction of differentiation. Adipogenesis within each of the scaffolds was investigated through gene expression analysis using end point and real time reverse transcriptase polymerase chain reaction (RT-PCR). The results indicate that the cell-adhesive PDM scaffolds facilitated proliferation and viability, while differentiation was augmented when the cells were encapsulated in the non-adhesive XLHA gels.

[Image: see text] In this work we investigate the feasibility of modifying porcine-derived BAM to include HA with a view to developing a model, artificial extracellular matrix for the study of bladder cell-matrix interactions. HA-DPTH was incorporated into BAM disks and then cross-linked oxidatively to a disulfide containing hydrogel. Disks were seeded with bladder smooth muscle cells (BSMC) and UEC under three culture configurations and incubated for 3, 7, and 14 d. At each time point, matrix contraction was measured, and media supernatants assayed for cell-secreted gelatinase activity. To evaluate cell adherence and organization, triple immunofluorescent labeling of cell nuclei, actin cytoskeleton, and focal contacts was performed. HA-modified BAM exhibited a significant increase in matrix contraction and induced a higher level of cell-secreted gelatinase activity compared to unmodified BAM. Immunofluorescent labeling demonstrated that BSMCs remained adherent to both scaffold types over time. The distribution and organization of the cytoskeleton and focal contacts did not appear to be altered by the presence of HA. Interestingly, cellular infiltration into modified BAM was evident by 7 d and continued beyond 14 d, while BSMCs seeded onto unmodified BAM remained localized to the surface out to 14 d, with minimal infiltration evident only at day 28. These differences in cell infiltration support the gelatinase activity results. Increases in cell migration and matrix proteolysis in the presence of HA may be contributing factors toward BAM remodeling leading to increased matrix contraction with time. The model ECM developed in this work will be utilized for future studies aimed at elucidating the mechanisms controlling key remodeling events associated with bladder repair. Matrix contraction of cell-seeded BAM scaffolds.

Bladder acellular matrix (BAM) is a promising candidate for urinary biomaterials development. In the current work we have modified the BAM construct to include two biologically active components; hyaluronan (HA) and a peptide (SP4.2) derived from secreted protein, acidic, rich in cysteine (SPARC), a matricellular glycoprotein. In order to assess the potential of an HA/SP4.2 modified BAM to influence cellular functions associated with bladder healing, experiments were conducted to evaluate the individual and combined effects of these molecules on in vitro fibroproliferative endpoints within a co-culture model. Thiol-modified HA (246 kDa, 15 mg/ml)+/-SP4.2 (200 microm) was incorporated and cross-linked into BAM disks through disulfide bond formation. The following scaffolds compositions were then evaluated in a bladder smooth muscle cell (SMC)-urothelial (UEC) cell co-culture model: BAM unmodified; BAM+HA, BAM+SP4.2 (media addition); BAM+HA+SP4.2 (media addition); BAM+HA+SP4.2 (matrix incorporated). At 3, 7 and 14 days post-seeding, SMC-mediated matrix contraction and gelatinolytic activity were evaluated. HA-modified BAM exhibited a significantly higher degree of contraction and gelatinase activity compared to unmodified BAM. In contrast, addition of SP4.2 to BAM produced a negligible effect on contraction, while significantly reducing gelatinase activity. Matrices containing both molecules displayed significant increases in contraction, while gelatinase activity was dependent upon the method of peptide delivery. These results demonstrate that both HA and SP4.2 have significant, yet distinct effects on the contractile and proteolytic activity of bladder SMCs and suggest that a modified BAM may be capable of modulating processes associated with post-surgical graft contracture and scar formation.

The objective of this study was to evaluate the ability of bladder acellular matrix (BAM) to support the individual and combined growth of primary porcine bladder smooth muscle (SMC) and urothelial (UEC) cells. An in vitro co-culture system was devised to evaluate the effect of UEC on (i) SMC-mediated contraction of BAM discs, and (ii) SMC invasiveness into BAM. Cells were seeded onto BAM discs under 4 different culture conditions. Constructs were incubated for 1, 7, 14 and 28 days. Samples were then harvested for evaluation of matrix contraction. Immunohistochemistry (IHC) was utilized to examine cellular organization within the samples and conditioned media supernatants analyzed for net gelatinase activity. BAM contraction was significantly increased with co-culture. The same side co-culture configuration lead to a greater reduction in surface area than opposite side co-culture. IHC revealed enhanced SMC infiltration into BAM when co-culture was utilized. A significant increase in net gelatinase activity was also observed with the co-culture configuration. Enhanced infiltration and contractile ability of bladder SMCs with UEC co-culture may, in part, be due to an increase in gelatinase activity. The influence of bladder UECs on SMC behaviour in vitro indicates that BAM may contain some key inductive factors that serve to promote important bladder cell-cell and cell-matrix interactions.

To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation, a common approach to mimic some of the spatial and temporal aspects of in vivo development, are affected by three factors: input hESC composition, input hESC colony size, and EB size. Using a microcontact printing approach, size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently, size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover, upon forming EBs from these MP-hESCs, we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MPEBs generated from endoderm-biased (high Gata6/Pax6) input hESCs, higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely, neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size, typical in most differentiation strategies, produces subsets of appropriate conditions for differentiation into specific cell types. Moreover, our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories. [ABSTRACT FROM AUTHOR]

Abstract: A tissue-engineered adipose substitute would have numerous applications in plastic and reconstructive surgery. This work involves the characterization of the in vitro cellular response of primary human adipose-derived stem cells (ASC) to three dimensional, naturally derived scaffolds. To establish a more thorough understanding of the influence of the scaffold environment on ASC, we have designed several different soft tissue scaffolds composed of decellularized human placenta and crosslinked hyaluronan (XLHA). The cellular organization within the scaffolds was characterized using confocal microscopy. Adipogenic differentiation was induced and the ASC response was characterized in terms of glycerol-3-phosphate dehydrogenase (GPDH) activity and intracellular lipid accumulation. The results indicate that the scaffold environment impacts the ASC response and that the adipogenic differentiation of the ASC was augmented in the non-adhesive XLHA gels. [Copyright &y& Elsevier]