Fabrication of tissue engineering scaffolds with spatial control over architecture and cell-matrix interactions in 3D

Abstract

The key accomplishment of this work is the demonstration of spatial control over architecture and surface chemistry in three-dimensional tissue engineering scaffolds. Tissues are characterized by a well-defined three-dimensional arrangement of cells. Spatial control of scaffold elements may be used to encourage the organization of cells into conformations resembling those of native tissue. Patterned scaffolds can be used to explore the healing process and then to design scaffolds with improved healing properties. Patterned architectures were fabricated from hydroxyapatite (HA), biodegradable polyesters (PLLA & PLLGA). and composites of degradable polyesters with bone (rat, bovine & human) using the Three-Dimensional Printing™ process. Two extremes in scaffold design were explored: l) dense structures for strength but with large (600 μm) channels for tissue and vasculature ingrowth. and 2) porous structures with room for cell attachment and growth. Porous structures fabricated from PLLGA and rat bone were implanted subcutaneously on the backs of rats. A typical inflammatory response was observed indicating an acceptable level of biocompatibility for 3DPTM fabricated devices. Dense PLLGA devices fabricated by 3DP™ were shown to still contain significant amounts of chloroform (-5 wt%) after conventional vacuum drying. Liquid C02 extraction was demonstrated to be capable of reducing chloroform in these devices to levels below 50 ppm. Drying was modeled as a diffusion process and diffusion coefficients were estimated for both a batch and a continuous-flow extraction system as 2.47x 10·4 and 3. 18 x10-4 cm2/min. respectively. The model predicts that 1.5 and 9 hours of extraction are needed to reach chloroform levels of <50 ppm in l & 3 mm thick PLLGA bars. respectively. Scaffolds with patterned surface chemistry were fabricated by printing Pluronic® F 127. a surfactant molecule containing PEO chains, in selected locations. Spatial control of MG-63 cell adhesion and morphology was demonstrated on patterned PLLGA surfaces and porous scaffolds. Cell numbers were reduced on Pluronic® modified regions and those attached were less spread and present only in the lower regions of the scaffold. The MG-63 osteosarcoma derived cell line was used to develop assays for measuring cell adhesion. differentiation. and migration in 30 scaffolds. The adhesion, migration and differentiation of rat osteoblasts was then systematically analyzed on nonpatterned scaffolds fabricated with different concentrations of Pluronic® (0, 0.0 I, 0.1 & 0.5% ). Adhesion and migration of rat osteoblasts decreased with increasing Pluronic® concentration. Although measurements were not statistically different. differentiation was judged to increase with Pluronic® concentration because proliferation decreased, alkaline phosphatase activity increased. and cells appeared less fibroblastic and had more microvilli. No significant differences in rat oste0blast behavior were seen on patterned scaffolds fabricated by printing one side with 0.5% Pluronic®. The hypothesis that Pluronic® migrates to the non-Pluronic® side is supported by the fact that Pluronic® is present in the washes generated during the salt-leaching step of fabrication.