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A significant challenge in tissue engineering is the creation of tissues with stratified morphology or embedded microstructures. This study investigated methods to fabricate composite gels from separately deposited alginate layers and examined the effects of processing methods on the mechanics of adhesion. Laminated alginate gels were created through a three step process which included: treatment of the interfaces with citrate; annealing of the gels to allow for molecular rearrangement of the alginate chains; and exposure to a CaCl(2) to crosslink the alginate sheets. Process variables included volume and concentration of applied citrate, annealing time, incubation time in CaCl(2), and CaCl(2) concentration. Laminated sheets were tested in lap-shear geometry to characterize failure phenomena and mechanical properties. The site of failure within the gel depended on the integrity of the interface, with weaker gels delaminating and gels with mechanical properties similar to that of bulk gels failing randomly throughout the thickness. Citrate volume, citrate concentration, CaCl(2) incubation time, and CaCl(2) concentration altered the mechanical properties of the laminated alginate sheets, while annealing time had little effect on all measured parameters. This study demonstrates the integration of separately fabricated alginate layers to create mechanically or chemically anisotropic or heterogeneous structures., (Copyright 2007 Wiley Periodicals, Inc.)

Motivated by the necessity to engineer appropriately stratified cartilage, the shear mechanics of layered, bovine chondrocyte-seeded 20mg/mL alginate scaffolds were investigated and related to the structure and biochemical composition. Chondrocyte-seeded alginate scaffolds were exposed to a calcium-chelating solution, layered, crosslinked in CaCl(2), and cultured for 10 weeks. The shear mechanical properties of the layered gels were statistically similar to those of the non-layered controls. Shear modulus of layered gels increased by approximately six-fold while toughness and shear strength increased by more than two-fold during the culture period. Hydroxyproline content in both layered gels and controls had statistically significant increases after 6 weeks. Glycosaminoglycan (GAG) content of controls increased throughout culture while GAG content in layered gels leveled off after 4 weeks. Hematoxylin and eosin histological staining showed tissue growth at the interface over the first 4 weeks. Shear mechanical properties in the engineered tissues showed significant correlations to hydroxyproline content. Dependence of interfacial mechanical properties on hydroxyproline content was most evident for layered gels when compared to controls, especially for toughness and shear strength. Additionally, interfacial properties showed almost no dependence on GAG content. These findings demonstrate the feasibility of creating stratified engineered tissues through layering and that collagen deposition is necessary for interfacial integrity.