Anna Giovanna Sciancalepore

Microvascular endothelial cell spreading and proliferation on nanofibrous scaffolds by polymer blends with enhanced wettability

The objective of this study is elucidating the mechanisms by which the wettability of nanofibrous electrospun mats varies in polymer blends, and highlighting how this can play a pivotal role in enhancing the viability of cultured microvascular endothelial cells (EC). A functional microvascular network is essential for supplying bioengineered tissues with oxygen and nutrients while removing metabolic wastes. An in vitro pre-vascularization strategy consists of seeding EC on scaffolds, which in turn promotes cells infiltration, adhesion and functionality. We use electrospun poly-L-lactic acid (PLLA) and gelatin (Gel) as prototype materials for realizing nanofibrous scaffolds as bioartificial architectures to improve the proliferation and the functionality of human microvascular ECs (HMEC-1). HMEC-1 seeded on electrospun scaffolds adhere, remain viable, proliferate and positively express the endothelial cell marker CD31 particularly on blend PLLA/Gel fibers, which exhibit wettability enhanced with respect to both the constituent polymers, and are therefore especially promising constructs for promoting the formation of functional endothelial tissue. The wettability characteristics of the blend polymer fibrous scaffolds are modeled and discussed. These results can be valuable for the future design of pre-vascularized scaffolds with enhanced wettability properties for functional tissue engineered implants, with ECs able to form in perspective an effectively functioning vasculature upon implantation.

Combined Nano‐ and Micro‐Scale Topographic Cues for Engineered Vascular Constructs by Electrospinning and Imprinted Micro‐Patterns

The major cause of synthetic vessel failure is thrombus and neointima formation. To prevent these problems the creation of a continuous and elongated endothelium inside lumen vascular grafts might be a promising solution for tissue engineering. Different micro- and nano-surface topographic cues including grooved micro-patterns and electrospun fibers have been previously demonstrated to guide the uniform alignment of endothelial cells (ECs). Here, with a very simple and highly versatile approach we combined electrospinning with soft lithography to fabricate nanofibrous scaffolds with oriented fibers modulated by different micro-grooved topographies. The effect of these scaffolds on the behavior of the ECs are analyzed, including their elongation, spreading, proliferation, and functioning using unpatterned random and aligned nanofibers (NFs) as controls. It is demonstrated that both aligned NFs and micro-patterns effectively influence the cellular response, and that a proper combination of topographic parameters, exploiting the synergistic effects of micro-scale and sub-micrometer features, can promote EC elongation, allowing the creation of a confluent ECs monolayer in analogy with the natural endothelium as assessed by the positive expression of vinculin. Combining different micro- and nano-topographic cues by complementary soft patterning and spinning technologies could open interesting perspectives for engineered vascular replacement constructions.

Rapid nested-PCR for tyrosinase gene detection on chip

The availability of non-invasive, fast and sensitive technologies for detection of circulating cancer cells is still a critical need of clinical oncology, particularly for diagnosis of aggressive and highly metastatic tumors, like malignant melanoma. Here we present the first nested polymerase chain reaction process carried out by a microfabricated, hybrid plastic-glass microfluidic chip on the tyrosinase gene, a predictive marker for melanoma diagnosis. The device is a hybrid system consisting of a glass microchannel embedded in an elastomeric matrix, and operating in flow-oscillating modality on a droplet of biological sample. The convection heat transfer and the temperature distribution inside the carrier fluid in the device are investigated. The oil responds to temperature changes with a characteristic time around 53 s, and exhibits three different thermal gradients along the capillary, with temperature variations below 4°C in correspondence of heater electrodes. The sample heating/cooling rates in the chip are as high as 16°C/s, allowing rapid processes. The nested polymerase chain reaction process is performed in less than 50 min, namely more than four times faster than in a standard thermocycler. The rapidity of the analysis method, combined with the simple and low-cost fabrication, reduced sample evaporation, and flexibility of the overall microfluidic platform, make it promising for the detection of events of tumor spreading.

Reduction of water evaporation in polymerase chain reaction microfluidic devices based on oscillating-flow

Producing polymeric or hybrid microfluidic devices operating at high temperatures with reduced or no water evaporation is a challenge for many on-chip applications including polymerase chain reaction (PCR). We study sample evaporation in polymeric and hybrid devices, realized by glass microchannels for avoiding water diffusion toward the elastomer used for chip fabrication. The method dramatically reduces water evaporation in PCR devices that are found to exhibit optimal stability and effective operation under oscillating-flow. This approach maintains the flexibility, ease of fabrication, and low cost of disposable chips, and can be extended to other high-temperature microfluidic biochemical reactors.

Muscle unloading potentiates the effects of acetyl‐L‐carnitine on the slow oxidative muscle phenotype

The effect of acetyl‐L‐carnitine (ALCAR) supplementation to 3‐month‐old rats in normal‐loading and unloading conditions has been here investigated by a combined morphological, biochemical and transcriptional approach to test whether ALCAR might cause a remodeling of the metabolic/contractile phenotype of soleus muscle. Morphological assessment demonstrated an increase of type I oxidative fiber content and cross‐sectional area in ALCAR‐treated animals both in normal‐loading and in unloading conditions. ALCAR prevented loss of mitochondrial mass in unloaded animals whereas no ALCAR‐dependent increase of mitochondrial mass occurred in normal‐loaded muscle. Validated microarray analysis delineated an ALCAR‐induced maintenance of a slow‐oxidative expression program only in unloaded soleus muscle. Indeed, the muscle adjustment of the expression profile of factors underlying mitochondrial oxidative metabolism, protein turnover, fiber type differentiation and an adaptation of voltage‐gated ion channel expression was distinguishable with respect to the loading status. This selectivity may suggest a key role of muscle loading status in the manifestation of ALCAR effects. The results extend to a broader level of biological informations the previous notion on ALCAR positive effect in rat soleus muscle during unloading and point to a role of ALCAR for the maintenance of its slow‐oxidative fiber character.

Correction: A Bioartificial Renal Tubule Device Embedding Human Renal Stem/Progenitor Cells

There are errors in Figure 3, panels E, F, and G. These panels were reproduced from Figure 1 of Sallustio F, Serino G, Costantino V, Curci C, Cox SN, et al. (2013) miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells. PLOS ONE 8(7): e68296. doi:10.1371/journal.pone.0068296 Additionally, the cytofluorimetric images in Figure 3, panels B (CD133) and D (CD44) are the same respectively as Figure 1, panels A and C in Sallustio et al. PLOS ONE (2013). These panels represent the same cells in each article. An investigation by a Scientific Committee at the University of Bari concluded that these image duplications arose by inadvertent error, due to poor management of the image archive, and that this does not affect the results and conclusions. The authors have provided the correct image files for Figure 3, panels E, F, and G in S1 File. A correction is also being made to Sallustio et al. PLOS ONE (2013). Fig 3. Fulfillment of a bioartificial proximal tubule on-a-chip embedding ARPCs. (A) Scheme of the glomerulus and the proximal tubule structure in a human kidney nephron. Here tubular ARPCs were seeded into the device, whose cross section illustrates a confluent layer of ARPCs within the lumen microchannel and adherent to the membrane. After 4 days of culture, the lumen microchannel was perfused with the complete culture medium containing urea (UR) and creatinine (CR) and the medium without UR and CR was injected in counter-current into the lower, interstitial microchannel. (B–G) Characterization of isolated tubular ARPCs. Cytofluorimetric analysis shows the expression of CD133 (B), CD24 (C), CD44 (D). Immunofluorescence detection evidences the expression of Oct-4 (E), PAX-2 (F), BMI-1 (G). Scale bars = 50 μm. (H–I) Confluent growth of ARPCs in the device attested by immunostaining of cells with DAPI (blue) (H) and TRITC-phalloidin (red) (I). Scale bars = 100 μm.