Enhancing the Three-Dimensional Structure of Adherent Gingival Fibroblasts and Spheroids via a Fibrous Protein-Based Hydrogel Cover.

Tissue engineering-based therapies rely on the delivery of monolayered fibroblasts on two-dimensional polystyrene-coated and extracellular matrix (ECM) surfaces to regenerate connective tissues. However, this approach may fail to mimic their three-dimensional (3D) native architecture and function. We hypothesize that ECM fibrous proteins, which direct the migration of cells in vivo, may attach and guide polystyrene- and Matrigel™-ECM (M-ECM)-adherent fibroblasts to rearrangement into large multicellular macrostructures with the ability to proliferate. Gingival monolayered fibroblasts and their derived spheroids were added and adhered to tissue culture polystyrene and M-ECM surfaces. The cells were covered with a layer of collagen1 hydrogel combined with vitronectin, fibronectin or fibrin, or 10% M-ECM. The development of 3D cell constructs was characterized by epifluorescence and confocal scanning microscope image analysis. The ECM turnover and the proliferative capabilities of the fibroblasts were determined via gene expression profiling of collagen1, fibronectin, matrix metalloproteinase/metallopeptidase 2, Nanog, and SRY (sex-determining region Y)-box2 (Sox2). Expression of the Sox2 protein was followed by immunostaining. The collagen1 protein had the strongest effect on monolayered and spheroid cell rearrangements, forming large spherical shapes and fused 3D macroconstructs. The addition of fibrin protein was typically required to achieve a similar effect on M-ECM-adherent monolayered fibroblasts. The spheroid fusion process was followed by an increase in cell density and the formation of tight clusters. The fused spheroids continued to maintain their intracellular ECM turnover and proliferation capacities. Collagen1 is a valuable component in the rearrangement of adherent fibroblast monolayers and spheroids. Fibroblast spheroids should preferably be used as basic building blocks to assemble multicellular connective tissue-like macrostructures.

Blow-spun chitosan/PEG/PLGA nanofibers as a novel tissue engineering scaffold with antibacterial properties.

Blow spinning is continuing to gain attention in tissue engineering, as the resultant nanofibrous structures can be used to create a biomimetic environment. In this study, blow spinning was used to construct nanofiber scaffolds with up to 10 % chitosan and poly(DL-lactide-co-glycolide) in the absence or presence of poly(ethylene glycol). Scanning electron microscopy demonstrated that nanofibers were distributed randomly to form three-dimensional mats. With respect to chitosan concentration, the average fiber diameter did not differ statistically in either the absence or presence of poly(ethylene glycol). In poly(ethylene glycol)-formulations, the average fiber diameter ranged from (981.9 ± 611.3) nm to (1139.2 ± 814.2) nm. In vitro cellular metabolic activity and proliferation studies using keratinized rat squamous epithelial cells (RL-65) showed that cytocompatibility was not compromised with the addition of poly(ethylene glycol). The cell responses at lower (1 and 2.5 %) chitosan concentrations were not significantly different from the groups without chitosan or no scaffold when cultivated for 3, 6, or 9 days. However, >15 % reduction in cellular responses were observed at 10 % chitosan. In presence of poly(ethylene glycol), nearly a 1-log incremental reduction in the number of colony forming units of Streptococcus mutans occurred as the chitosan concentration increased from 0-1 to 2.5 %. Bacterial preparations tested with poly(ethylene glycol) and 5 or 10 % chitosan were not significantly different than the positive kill control. Taken together, the most favorable conditions for attaining cytocompatibility and maintaining antibacterial functionality existed in poly(ethylene glycol)/poly(DL-lactide-co-glycolide) blow-spun scaffolds with integrated 1 or 2.5 % chitosan.

The chemical and physical characteristics of single-walled carbon nanotube film impact on osteoblastic cell response.

Carbon-nanotube-based substrates have been shown to support the growth of different cell types and, as such, have raised considerable interest in view of their possible use in biomedical applications. Nanotube matrices are embedded in polymers which cause inherent changes in nanotube chemical and physical film properties. Thus, it is critical to understand how the physical properties of the film affect the biology of the host tissue. Here, we investigated how the physical and chemical properties of single-walled carbon nanotubes (SWNT) films impact the response of MC3T3-E1 bone osteoblasts. We found that two fundamental steps in cell growth-initial attachment to the substrate and proliferation-are strongly dependent on, respectively, the energy and roughness of the surface. Thus, fine-tuning the properties of the film may represent a valid strategy to optimize the response of the biological host.

Toxicity induced enhanced extracellular matrix production in osteoblastic cells cultured on single-walled carbon nanotube networks.

A central effort in biomedical research concerns the development of materials for sustaining and controlling cell growth. Carbon nanotube based substrates have been shown to support the growth of different kinds of cells (Hu et al 2004 Nano Lett. 4 507-11; Kalbacova et al 2006 Phys. Status Solidi b 13 243; Zanello et al 2006 Nano Lett. 6 562-7); however the underlying molecular mechanisms remain poorly defined. To address the fundamental question of mechanisms by which nanotubes promote bone mitosis and histogenesis, primary calvariae osteoblastic cells were grown on single-walled carbon nanotube thin film (SWNT) substrates. Using a combination of biochemical and optical techniques we demonstrate here that SWNT networks promote cell development through two distinct steps. Initially, SWNTs are absorbed in a process that resembles endocytosis, inducing acute toxicity. Nanotube-mediated cell destruction, however, induces a release of endogenous factors that act to boost the activity of the surviving cells by stimulating the synthesis of extracellular matrix.

Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissue regeneration.

Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by ImageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within three days of incubation, fibroblast spheroids interacted with the fibers, and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for engineering soft and dense connective tissues with the required structural characteristics and functions needed for wound healing applications. Rapid regeneration of these layers should enhance healing of open wounds in a harsh oral environment.

Bioactive Polymeric Materials for Tissue Repair.

Bioactive polymeric materials based on calcium phosphates have tremendous appeal for hard tissue repair because of their well-documented biocompatibility. Amorphous calcium phosphate (ACP)-based ones additionally protect against unwanted demineralization and actively support regeneration of hard tissue minerals. Our group has been investigating the structure/composition/property relationships of ACP polymeric composites for the last two decades. Here, we present ACP's dispersion in a polymer matrix and the fine-tuning of the resin affects the physicochemical, mechanical, and biological properties of ACP polymeric composites. These studies illustrate how the filler/resin interface and monomer/polymer molecular structure affect the material's critical properties, such as ion release and mechanical strength. We also present evidence of the remineralization efficacy of ACP composites when exposed to accelerated acidic challenges representative of oral environment conditions. The utility of ACP has recently been extended to include airbrushing as a platform technology for fabrication of nanofiber scaffolds. These studies, focused on assessing the feasibility of incorporating ACP into various polymer fibers, also included the release kinetics of bioactive calcium and phosphate ions from nanofibers and evaluate the biorelevance of the polymeric ACP fiber networks. We also discuss the potential for future integration of the existing ACP scaffolds into therapeutic delivery systems used in the precision medicine field.

Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells.

Recent work demonstrates that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the three-dimensional (3D) structure of nanofibers. We hypothesized that nanofiber effects on cell behavior may be mediated by changes in organelle structure and function. To test this hypothesis, human bone marrow stromal cells (hBMSCs) were cultured on poly(ε-caprolactone) (PCL) nanofibers scaffolds and on PCL flat spuncoat films. After 1 day-culture, hBMSCs were stained for actin, nucleus, mitochondria, and peroxisomes, and then imaged using 3D confocal microscopy. Imaging revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of cell volume during culture on nanofibers than on films, suggesting enhancement of the nuclear and peroxisomal functional capacity. Organelles adopted morphologies with greater 3D-character on nanofibers, where the Z-Depth (a measure of cell thickness) was increased. Comparisons of organelle positions indicated that the nucleus, mitochondria, and peroxisomes were closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, hBMSC bioassay measurements (DNA, peroxidase, bioreductive potential, lactate, and adenosine triphosphate (ATP)) indicated that peroxidase activity may be enhanced during nanofiber culture. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure and positioning, which may affect organelle functional capacity and transport. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. J Biomed Mater Res Part B: Appl Biomater, 2016. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 989-1001, 2017.

Universal electronic-cigarette test: physiochemical characterization of reference e-liquid.

BACKGROUND: Despite the rising health and safety concerns of e-cigarettes, a universal e-cigarette testing method is still in its early developmental stage. The aim of this study was to develop an e-liquid Reference Material that can be used to improve accuracy and reproducibility of research results, and advance health risk assessment of e-cigarette products. METHODS: E-liquid Reference Material was developed by purity assessment, gravimetric measurement, homogeneity testing, and stability testing with material and instrument traceability (adopted from ISO 35:2006E). RESULTS: Homogeneity tests showed e-liquid Reference Material requires ≥ 1 h rotation at a speed of 5 rpm to reach complete homogeneity. Stability tests showed homogeneity is intact for at least 2 weeks without secondary separation, and e-liquids are stable in 21 °C-50 °C thermocycling conditions up to 72 h. A change in the e-liquid color was first observed at day seven, and progressed to 2- and 16 - fold increase in absorbance by one and 6 months respectively. We found that e-liquids do not have inherent material instabilities such as immiscibility or secondary separation. However, discrepancies in concentration and composition arose mainly due to viscosity of propylene glycol and glycerin. Aerosol generated from the e-liquid Reference Material had 16 chemical-byproducts and was composed of ~634,000 particles of which 38% were Fine Particulate Matters (<0.5 µm in diameter). CONCLUSIONS: The efforts described here to create a standardized e-liquid Reference Material aim to provide unbiased and robust testing parameters that may be useful for researchers, the industry and government agencies. Additionally, the reference e-liquid could open a channel of conversation among different laboratories by providing the means of independent verification and validation while establishing a system of transparency and reproducibility in materials and methods.

Rapid fabrication of poly(DL-lactide) nanofiber scaffolds with tunable degradation for tissue engineering applications by air-brushing.

Polymer nanofiber based materials have been widely investigated for use as tissue engineering scaffolds. While promising, these materials are typically fabricated through techniques that require significant time or cost. Here we report a rapid and cost effective air-brushing method for fabricating nanofiber scaffolds using a simple handheld apparatus, compressed air, and a polymer solution. Air-brushing also facilities control over the scaffold degradation rate without adversely impacting architecture. This was accomplished through a one step blending process of high (M w ≈ 100 000 g mol(-1)) and low (M w ≈ 25 000 g mol(-1)) molecular weight poly(DL-lactide) (PDLLA) polymers at various ratios (100:0, 70:30 and 50:50). Through this approach, we were able to control fiber scaffold degradation rate while maintaining similar fiber morphology, scaffold porosity, and bulk mechanical properties across all of the tested compositions. The impact of altered degradation rates was biologically evaluated in human bone marrow stromal cell (hBMSC) cultures for up to 16 days and demonstrated degradation rate dependence of both total DNA concentration and gene regulation.

Airbrushed composite polymer Zr-ACP nanofiber scaffolds with improved cell penetration for bone tissue regeneration.

Electrospun polymer nanofibers have multiple applications in the tissue engineering field despite limited cell penetration within the scaffolds and slow synthesis rates. Airbrushing, a proposed alternative to traditional electrospinning, is a technique capable of synthesizing open structure nanofiber scaffolds at high rates. In this study, three biocompatible polymers-poly-D,L-lactic acid (P-DL-LA), polycaprolactone (PCL), and poly(methyl methacrylate) (PMMA), were airbrushed to form networks for bone tissue regeneration. All three polymers were loaded with up to 20% (w/w) zirconium-modified amorphous calcium phosphate (Zr-ACP). A simple one-step mix and straightforward material deposition yielded open structure networks with well-distributed Zr-ACP. Cell penetration within the airbrushed scaffolds was found to be more than twice the cell penetration within conventional electrospun networks. The airbrushed polymer network supported cell growth and differentiation. Cells grown on the Zr-ACP in P-DL-LA fibers exhibited improved levels of osteocalcin protein with an increase in the Zr-ACP content by day 16. This airbrushing method promises to be a viable and attractive alternative to currently used electrospinning techniques in the formation of composite 3D nanofiber scaffolds for tissue engineering applications.

In Situ Deposition of PLGA Nanofibers via Solution Blow Spinning.

Nanofiber mats and scaffolds have been widely investigated for biomedical applications. Commonly fabricated using electrospinning, nanofibers are generated ex situ using an apparatus that requires high voltages and an electrically conductive target. We report the use of solution blow spinning to generate conformal nanofiber mats/meshes on any surface in situ, utilizing only a commercial airbrush and compressed CO2. Solution and deposition conditions of PLGA nanofibers were optimized and mechanical properties characterized with dynamic mechanical analysis. Nanofiber mat degradation was monitored for morphologic and molecular weight changes in vitro. Biocompatibility of the direct deposition of nanofibers onto two cell lines was demonstrated in vitro and interaction with blood was qualitatively assessed with scanning electron microscopy. A pilot animal study illustrated the wide potential of this technique across multiple surgical applications, including its use as a surgical sealant, hemostatic, and buttress for tissue repair.

The support of bone marrow stromal cell differentiation by airbrushed nanofiber scaffolds.

Nanofiber scaffolds are effective for tissue engineering since they emulate the fibrous nanostructure of native extracellular matrix (ECM). Although electrospinning has been the most common approach for fabricating nanofiber scaffolds, airbrushing approaches have also been advanced for making nanofibers. For airbrushing, compressed gas is used to blow polymer solution through a small nozzle which shears the polymer solution into fibers. Our goals were 1) to assess the versatility of airbrushing, 2) to compare the properties of airbrushed and electrospun nanofiber scaffolds and 3) to test the ability of airbrushed nanofibers to support stem cell differentiation. The results demonstrated that airbrushing could produce nanofibers from a wide range of polymers and onto a wide range of targets. Airbrushing was safer, 10-fold faster, 100-fold less expensive to set-up and able to deposit nanofibers onto a broader range of targets than electrospinning. Airbrushing yielded nanofibers that formed loosely packed bundles of aligned nanofibers, while electrospinning produced un-aligned, single nanofibers that were tightly packed and highly entangled. Airbrushed nanofiber mats had larger pores, higher porosity and lower modulus than electrospun mats, results that were likely caused by the differences in morphology (nanofiber packing and entanglement). Airbrushed nanofiber scaffolds fabricated from 4 different polymers were each able to support osteogenic differentiation of primary human bone marrow stromal cells (hBMSCs). Finally, the differences in airbrushed versus electrospun nanofiber morphology caused differences in hBMSC shape where cells had a smaller spread area and a smaller volume on airbrushed nanofiber scaffolds. These results highlight the advantages and disadvantages of airbrushing versus electrospinning nanofiber scaffolds and demonstrate that airbrushed nanofiber scaffolds can support stem cell differentiation.