Characterization of cell viability during bioprinting processes.

Bioprinting is an emerging technology in the field of tissue engineering and regenerative medicine. The process consists of simultaneous deposition of cells, biomaterial and/or growth factors under pressure through a micro-scale nozzle. Cell viability can be controlled by varying the parameters like pressure and nozzle diameter. The process itself can be a very useful tool for evaluating an in vitro cell injury model. It is essential to understand the cell responses to process-induced mechanical disturbances because they alter cell morphology and function. We carried out analysis and quantification of the degree of cell injury induced by bioprinting process. A parametric study with different process parameters was conducted to analyze and quantify cell injury as well as to optimize the parameters for printing viable cells. A phenomenological model was developed correlating the percentage of live, apoptotic and necrotic cells to the process parameters. This study incorporates an analytical formulation to predict the cell viability through the system as a function of the maximum shear stress in the system. The study shows that dispensing pressure has a more significant effect on cell viability than the nozzle diameter. The percentage of live cells is reduced significantly (by 38.75%) when constructs are printed at 40 psi compared to those printed at 5 psi.

Initial evaluation of vascular ingrowth into superporous hydrogels.

There is a need for new materials and architectures for tissue engineering and regenerative medicine. Based upon our recent results developing novel scaffold architecture, we hypothesized that this new architecture would foster vascularization, a particular need for tissue engineering. We report on the potential of superporous hydrogel (SPH) scaffolds for in vivo cellular infiltration and vascularization. Poly(ethylene glycol) diacrylate (PEGDA) SPH scaffolds were implanted in the dorsum of severe combined immunodeficient (SCID) mice and harvested after 4 weeks of in vivo implantation. The SPHs were visibly red and vascularized, as apparent when compared to the non-porous hydrogel controls, which were macroscopically avascular. Host cell infiltration was observed throughout the SPHs. Blood cells and vascular structures, confirmed through staining for CD34 and smooth muscle alpha-actin, were observed throughout the scaffolds. This novel soft material may be utilized for cell transplantation, tissue engineering and in combination with cell therapies. The neovasularization and limited fibrotic response suggest that the architecture may be conducive to cell survival and rapid vessel development.

Fabrication of three-dimensional polycaprolactone/hydroxyapatite tissue scaffolds and osteoblast-scaffold interactions in vitro.

Computer-aided tissue-engineering approach was used to develop a novel precision extrusion deposition (PED) process to directly fabricate Polycaprolactone (PCL) and composite PCL/hydroxyapatite (PCL-HA) tissue scaffolds. The process optimization was carried out to fabricate both PCL and PCL-HA (25% concentration by weight of HA) with a controlled pore size and internal pore structure of the 0 degrees /90 degrees pattern. Two groups of scaffolds having 60% and 70% porosity and with pore sizes of 450 and 750 microm, respectively, were evaluated for their morphology and compressive properties using scanning electron microscopy (SEM) and mechanical testing. Our results suggested that inclusion of HA significantly increased the compressive modulus from 59 to 84 MPa for 60% porous scaffolds and from 30 to 76 MPa for 70% porous scaffolds. In vitro cell-scaffolds interaction study was carried out using primary fetal bovine osteoblasts to assess the feasibility of scaffolds for bone tissue-engineering application. The cell proliferation and differentiation were calculated by Alamar Blue assay and by determining alkaline phosphatase activity. The osteoblasts were able to migrate and proliferate over the cultured time for both PCL as well as PCL-HA scaffolds. Our study demonstrated the viability of the PED process to the fabricate PCL and PCL-HA composite scaffolds having necessary mechanical property, structural integrity, controlled pore size and pore interconnectivity desired for bone tissue engineering.

Co-electrospun poly(lactide-co-glycolide), gelatin, and elastin blends for tissue engineering scaffolds.

In this study, we describe composite scaffolds composed of synthetic and natural materials with physicochemical properties suitable for tissue engineering applications. Fibrous scaffolds were co-electrospun from a blend of a synthetic biodegradable polymer (poly(lactic-co-glycolic acid), PLGA, 10% solution) and two natural proteins, gelatin (denatured collagen, 8% solution) and alpha-elastin (20% solution) at ratios of 3:1:2 and 2:2:2 (v/v/v). The resulting PLGA-gelatin-elastin (PGE) fibers were homogeneous in appearance with an average diameter of 380 +/- 80 nm, which was considerably smaller than fibers made under identical conditions from the starting materials (PLGA, 780 +/- 200 nm; gelatin, 447 +/- 123 nm; elastin, 1060 +/- 170 nm). Upon hydration, PGE fibers swelled to an average fiber diameter of 963 +/- 132 nm, but did not disintegrate. Importantly, PGE scaffolds were stable in an aqueous environment without crosslinking and were more elastic than those made of pure elastin fibers. To investigate the cytocompatibility of PGE, we cultured H9c2 rat cardiac myoblasts and rat bone marrow stromal cells (BMSCs) on fibrous PGE scaffolds. We found that myoblasts grew equally as well or slightly better on the scaffolds than on tissue-culture plastic. Microscopic evaluation confirmed that myoblasts reached confluence on the scaffold surfaces while simultaneously growing into the scaffolds. Histological characterization of the PGE constructs indicated that BMSCs penetrated into the center of scaffolds and began proliferating shortly after seeding. Our results suggest that fibrous scaffolds made of PGE and similar biomimetic blends of natural and synthetic polymers may be useful for engineering soft tissues, such as heart, lung, and blood vessels.

Carbon nanotube reinforced Bombyx mori silk nanofibers by the electrospinning process.

Nanocomposite fibers of Bombyx mori silk and single wall carbon nanotubes (SWNT) were produced by the electrospinning process. Regenerated silk fibroin dissolved in a dispersion of carbon nanotubes in formic acid was electrospun into nanofibers. The morphology, structure, and mechanical properties of the electrospun nanofibers were examined by field emission environmental scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and microtensile testing. TEM of the reinforced fibers shows that the single wall carbon nanotubes are embedded in the fibers. The mechanical properties of the SWNT reinforced fiber show an increase in Young's modulus up to 460% in comparison with the un-reinforced aligned fiber, but at the expense of the strength and strain to failure.

Electrospun protein fibers as matrices for tissue engineering.

Electrospinning has recently emerged as a leading technique for generating biomimetic scaffolds made of synthetic and natural polymers for tissue engineering applications. In this study, we compared collagen, gelatin (denatured collagen), solubilized alpha-elastin, and, as a first, recombinant human tropoelastin as biopolymeric materials for fabricating tissue engineered scaffolds by electrospinning. In extending previous studies, we optimized the shape and size (diameter or width) of the ensuing electrospun fibers by varying important parameters of the electrospinning process, such as solute concentration and delivery rate of the polymers. Our results indicate that the average diameter of gelatin and collagen fibers could be scaled down to 200-500 nm without any beads, while the alpha-elastin and tropoelastin fibers were several microns in width. Importantly, and contrary to any hitherto reported structures of electrospun polymers, fibers composed of alpha-elastin, especially tropoelastin, exhibited "quasi-elastic" wave-like patterns at increased solution delivery rates. The periodicity of these wave-like tropoelastin fibers was partly affected by the delivery rate. Atomic force microscopy was utilized to profile the topography of individual electrospun fibers and microtensile testing was performed to measure their mechanical properties. Cell culture studies confirmed that the electrospun engineered protein scaffolds support attachment and growth of human embryonic palatal mesenchymal (HEPM) cells.

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils.

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.