An important issue in tissue engineering concerns the possibility of limited tissue ingrowth in tissue-engineered constructs because of insufficient nutrient transport. We report a dynamic flow culture system using high-aspect-ratio vessel rotating bioreactors and 3D scaffolds for culturing rat calvarial osteoblast cells. 3D scaffolds were designed by mixing lighter-than-water (density, <1 g͞ml) and heavier-than-water (density, >1 g͞ml) microspheres of 85:15 poly(lactide-co-glycolide). We quantified the rate of 3D flow through the scaffolds by using a particle-tracking system, and the results suggest that motion trajectories and, therefore, the flow velocity around and through scaffolds in rotating bioreactors can be manipulated by varying the ratio of heavier-than-water to lighter-than-water microspheres. When rat primary calvarial cells were cultured on the scaffolds in bioreactors for 7 days, the 3D dynamic flow environment affected bone cell distribution and enhanced cell phenotypic expression and mineralized matrix synthesis within tissue-engineered constructs compared with static conditions. These studies provide a foundation for exploring the effects of dynamic flow on osteoblast function and provide important insight into the design and optimization of 3D scaffolds suitable in bioreactors for in vitro tissue engineering of bone.
Agarose hydrogel scaffolds were engineered to stimulate and guide neuronal process extension in three dimensions in vitro. The extracellular matrix (ECM) protein laminin (LN) was covalently coupled to agarose hydrogel using the bifunctional cross-linking reagent 1,19- carbonyldiimidazole (CDI). Compared to unmodified agarose gels, LN-modified agarose gels significantly enhanced neurite extension from three-dimensionally (3D) cultured embryonic day 9 (E9) chick dorsal root ganglia (DRGs), and PC 12 cells. After incubation of DRGs or PC 12 cells with YIGSR peptide or integrin beta1 antibody respectively, the neurite outgrowth promoting effects in LN-modified agarose gels were significantly decreased or abolished. These results indicate that DRG/PC 12 cell neurite outgrowth promoting effect of LN-modified agarose gels involves receptors for YIGSR/integrin beta1 subunits respectively. 1,2-bis(10, 12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC)-based lipid microcylinders were loaded with nerve growth factor (NGF), and embedded into agarose hydrogels. The resulting trophic factor gradients stimulated directional neurite extension from DRGs in agarose hydrogels. A PC 12 cell-based bioassay demonstrated that NGF-loaded lipid microcylinders can release physiologically relevant amounts of NGF for at least 7 days in vitro. Agarose hydrogel scaffolds may find application as biosynthetic 3D bridges that promote regeneration across severed nerve gaps.
Understanding neural cell differentiation and neurite extension in three-dimensional scaffolds is critical for neural tissue engineering. This study explores the structure-function relationship between a 3D hydrogel scaffold and neural cell process extension and examines the role of ambient charge on neurite extension in 3D scaffolds. A range of agarose hydrogel concentrations was used to generate varied gel physical structures and the corresponding neurite extension was examined. Agarose gel concentration and the corresponding pore radius are important physical properties that influence neural cell function. The average pore radii of the gels were determined while the gel was in the hydrated state and in two different dehydrated states. As the gel concentration was increased, the average pore radius decreased exponentially. Similarly, the length of neurites extended by E9 chick DRGs cultured in agarose gels depends on gel concentration. The polycationic polysaccharide chitosan and the polyanionic polysaccharide alginate were used to incorporate charge into the 3D hydrogel scaffold, and neural cell response to charge was studied. Chitosan and alginate were covalently bound to the agarose hydrogel backbone using the bi-functional coupling agent 1,1'-carbonyldiimidazole. DRGs cultured in chitosan-coupled agarose gel exhibited a significant increase in neurite length compared to the unmodified agarose control. Conversely, the alginate-coupled agarose gels significantly inhibited neurite extension. This study demonstrates a strong, correlation between the ability of sensory ganglia to extend neurites in 3D gels and the hydrogel pore radius. In addition, our results demonstrate that charged biopolymers influence neurite extension in a polarity dependent manner.
The use of autografts for "bridging" peripheral nerve gaps is limited by lack of suitable donor nerve grafts. Using a tissue-engineering approach, we have designed a three-dimensional scaffold that presents laminin 1 (LN-1) and nerve growth factor (NGF) in vivo. Semipermeable polysulfone tubes were used as carriers to introduce the tissue-engineered scaffolds to a 10-mm sciatic nerve gap in adult rats. Two months after implantation, the gross morphology of the regenerated nerve, the success rate of regeneration, and the total number and density of myelinated axons in the tissue-engineered scaffolds matched that observed in autografts. LN-1- and NGF-containing scaffolds performed comparably to autografts when functional measures that include the relative gastrocnemius muscle weight and the sciatic functional index were quantified. Our results demonstrate that tissue-engineered scaffolds match the performance of autografts in an in vivo model of peripheral nerve regeneration, raising the possibility of the scaffolds being used clinically instead of scarce autografts.
Glial scar formation plays a critical role in the regenerative failure in the central nervous system of adult mammals through the formation of mechanical or biochemical barriers as a result of its molecular composition. In this study, we report an in vitro model to study growth-cone behavior at controlled 3D interfaces using layered agarose hydrogels. The behavior of growth cones from embryonic day 9 (E9) chick dorsal root ganglia (DRGs) at interfaces that were mismatched in terms of their elasticity or chondroitin sulfate content was quantitatively determined. A mechanical barrier formed by the elasticity mismatch of layered agarose gels greatly influenced the ability of neurites from E9 DRGs to cross the 3D interface. To form chondroitin sulfate-rich interfaces, chondroitin sulfate B was covalently coupled to agarose hydrogel. Compared with unmodified agarose gels, the presence of CS-B-modified agarose gels at the interface significantly inhibited E9 DRGs neurites. After treatment of CS-B-modified agarose gels with chondroitinase ABC, the inhibitory effects of CS-B at the interface were significantly decreased. The effect of doping CS-B gels with laminin 1 (LN-1)-coupled agarose gels was investigated as a potential strategy to overcome inhibitory interfaces. When CS-B agarose gels were doped with LN-1-coupled agarose gels, DRG neurite's ability to cross 3D interfaces was significantly enhanced compared with that of non-LN-1-containing interfaces presenting equivalent CS-B. Our in vitro model may be used to study the influence of individual components of glial scar on inhibition as well as to design strategies to overcome this inhibition.
Tissue engineering approaches for peripheral nerve regeneration employ biodegradable scaffolds coupled with growth factors for improved performance in regeneration of large nerve injuries. Electrospun nanofibers provide a versatile platform for fabrication of scaffolds with extracellular matrix like architecture and increased surface area. Incorporation of growth factors in nanofibers have been previously demonstrated using oil in water emulsion techniques but are associated with burst release and loss of valuable growth factor. Here, we show a novel blend of polycaprolactone and bovine serum albumin (BSA) to form nanofibers containing nerve growth factors. The BSA helps in overcoming the most common drawbacks associated with hydrophobic polymers such as reduced loading efficiency, long degradation periods, and burst release. The controlled release of nerve growth factor (NGF) from the nanofibers was evaluated using enzyme linked immune sorbent assay (ELISA) and PC12 based bioassay over a 28 day time period. A sustained release of NGF was obtained for at least 28 days. PC12 bioassays confirmed the bioactivity of the NGF, and showed that the released NGF was sufficient to induce neurite outgrowth from PC12 cells throughout the period of release, therefore, demonstrating the successful incorporation and controlled release potential of PCL BSA scaffolds.
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