Comparative analysis of poly-glycolic acid-based hybrid polymer starter matrices for in vitro tissue engineering

Biodegradable scaffold matrixes form the basis of any in vitro tissue engineering approach by acting as a temporary matrix for cell proliferation and extracellular matrix deposition until the scaffold is replaced by neo-tissue. In this context several synthetic polymers have been investigated, however a concise systematic comparative analyses is missing. Therefore, the present study systematically compares three frequently used polymers for the in vitro engineering of extracellular matrix based on poly-glycolic acid (PGA) under static as well as dynamic conditions. Ultra-structural analysis was used to examine the polymers structure. For tissue engineering (TE) three human fibroblast cell lines were seeded on either PGA-poly-4-hydroxybutyrate (P4HB), PGA-poly-lactic acid (PLA) or PGA-poly-caprolactone (PCL) patches. These patches were analyzed after 21days of culture qualitative by histology and quantitative by determining the amount of DNA, glycosaminoglycan and hydroxyproline. We found that PGA-P4HB and PGA-PLA scaffolds enhance tissue formation significantly higher than PGA-PCL scaffolds (p<0.05). Polymer remnants were visualized by polarization microscopy. In addition, biomechanical properties of the tissue engineered patches were determined in comparison to native tissue. This study may allow future studies to specifically select certain polymer starter matrices aiming at specific tissue properties of the bioengineered constructs in vitro

Combining dynamic stretch and tunable stiffness to probe cell mechanobiology in vitro.

Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G' = 0.3 kPa) to stiff (G' = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1 Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch

Engineering hybrid polymer-protein super-aligned nanofibers via rotary jet spinning

Cellular microenvironments are important in coaxing cells to behave collectively as functional, structured tissues. Important cues in this microenvironment are the chemical, mechanical and spatial arrangement of the supporting matrix in the extracellular space. In engineered tissues, synthetic scaffolding provides many of these microenvironmental cues. Key requirements are that synthetic scaffolds should recapitulate the native three-dimensional (3D) hierarchical fibrillar structure, possess biomimetic surface properties and demonstrate mechanical integrity, and in some tissues, anisotropy. Electrospinning is a popular technique used to fabricate anisotropic nanofiber scaffolds. However, it suffers from relatively low production rates and poor control of fiber alignment without substantial modifications to the fiber collector mechanism. Additionally, many biomaterials are not amenable for fabrication via high-voltage electrospinning methods. Hence, we reasoned that we could utilize rotary jet spinning (RJS) to fabricate highly aligned hybrid protein-polymer with tunable chemical and physical properties. In this study, we engineered highly aligned nanofiber constructs with robust fiber alignment from blends of the proteins collagen and gelatin, and the polymer poly-ε-caprolactone via RJS and electrospinning. RJS-spun fibers retain greater protein content on the surface and are also fabricated at a higher production rate compared to those fabricated via electrospinning. We measured increased fiber diameter and viscosity, and decreasing fiber alignment as protein content increased in RJS hybrid fibers. RJS nanofiber constructs also demonstrate highly anisotropic mechanical properties mimicking several biological tissue types. We demonstrate the bio-functionality of RJS scaffold fibers by testing their ability to support cell growth and maturation with a variety of cell types. Our highly anisotropic RJS fibers are therefore able to support cellular alignment, maturation and self-organization. The hybrid nanofiber constructs fabricated by RJS therefore have the potential to be used as scaffold material for a wide variety of biological tissues and organs, as an alternative to electrospinning

Average strain (± SD) within central region used for analysis of cell morphology for equibiaxial stretch (round loading post) and uniaxial stretch (Arctangle™ loading post).

<p>Average strain (± SD) within central region used for analysis of cell morphology for equibiaxial stretch (round loading post) and uniaxial stretch (Arctangle™ loading post).</p

Schematic of polyacrylamide gel on flexible silicone membrane under static (A) and stretched (B) conditions.

<p>Top view of a 22 mm diameter collagen-coated gel (∼70 µm thickness) is cast into a 35 mm diameter flexible-bottomed Flexcell™ well (C) and STREX well (C, insert). Image of Flexcell™ well (D) stretched above an Arctangle™ loading post and labeled with retroreflective beads for strain field analysis. Rectangle shows region analyzed in HDM software, arrows point to edge of gel. Scale bars = 10 mm in all panels.</p

hMSC response to stretch is unclear due to spreading on static soft gels.

<p>Micrographs of hMSCs cultured statically (left column) and following ∼10% cyclic equibiaxial strain (right column) for 6 hours on soft gels (0.3 kPa, top row) and stiff gels (50 kPa, bottom row). Staining for f-actin (green) and nuclei (blue) shows that hMSCs on soft gels (static and stretched) have unorganized actin fibers whereas cells on stiff gels have more organized actin fibers. Unlike VICs, hMSCs spread well on soft gels and stretch appears to increase the spread area of the cells slightly on stiff gels. Scale bar = 100 µm.</p

Strain field in region of interest is roughly uniform for pure uniaxial stretch.

<p>Strain maps for a soft gel (0.3 kPa) undergoing pure uniaxial strain in the X (A), Y (B), and XY (shear, C) directions demonstrating relatively homogenous strain and minimal shear within the area of analysis of cell morphology (dashed box). (D) CAD representation of the Arctangle™ loading platen over which the silicone membrane is stretched by vacuum pressure. Scale bars = 5 mm.</p

When cyclically stretched, cells on stiff substrates reduce spread area whereas cells on soft substrates increase spread area: Area (A) and perimeter (B) of VICs cultured on low (0.3 kPa) and high (50 kPa) stiffness gels subjected to 10% cyclic stretch at 1 Hz for 6 hours (grey bars) or static (black bars) culture.

<p>Shape factor (C) quantifies how rounded a cell is (a shape factor of 1 is perfectly circular, whereas a shape factor of 0 is extremely spread with many extensions). Cells of low and high shape factor are shown in C. Brackets above bars show significance between individual groups (two-way ANOVA, p<0.05).</p

Strain field in region of interest is roughly uniform for equibiaxial stretch.

<p>Strain maps for a soft gel (0.3 kPa) undergoing equibiaxial strain in the X (A), Y (B), and XY (shear, C) directions demonstrating relatively homogenous strain and minimal shear within the area of analysis of cell morphology (dashed box). (D) CAD representation of the circular loading platen over which the silicone membrane is stretched by vacuum pressure. Scale bars = 5 mm.</p

VICs on soft (0.3 kPa) and stiff (50 kPa) gels cultured under static and pure uniaxial stretch conditions (1 Hz, 10% stretch, 6 hrs).

<p>Cells cultured on soft substrates appear to have less realignment with stretch compared to the classic realignment perpendicular to the direction of stretch on the stiff substrates. Scale bar = 100 µm.</p