(616d) Spatially-Patterned Collagen-GAG Scaffolds for Regulating MSC Fate

The functional regeneration of multi-tissue structures such as the tendon-bone junction (TBJ) is a central challenge in modern tissue engineering that requires new technologies permitting simultaneous control of microstructural, mechanical, and biochemical properties in a spatially-defined manner. The TBJ is one of the most mechanically severe interfaces in the body, is a common site for injury, and current clinical strategies that rely on mechanical fixation rather than biological re-integration are inadequate. Collagen-glycosaminoglycan (CG) scaffolds are regulatory compliant extracellular matrix (ECM) analogs that have been applied to a variety of regenerative medicine challenges, most recently tendon tissue engineering [1]. Here we present an approach to spatially pattern microstructural (pore size and shape), mechanical, and biochemical (mineral content, biomolecule supplementation) properties within a single CG scaffold to drive spatially-defined multi-lineage mesenchymal stem cell (MSC) specification for TBJ regenerative applications.

TBJ scaffolds were fabricated by combining a directional solidification lyophilization technique [1] with a previously described liquid-phase co-synthesis method [2] to form scaffolds with a distinct zonal structure containing spatially-graded mineral content and pore anisotropy mimicking the native TBJ. Scaffolds supported long-term tenocyte (TC) and MSC viability for up to 8 weeks.  Anisotropic 3D scaffolds with larger pores and higher relative density were more mechanically competent and able to maintain aligned contact guidance cues, resulting in long term TC phenotypic stability with increased expression of scleraxis (15-fold) compared to 2D culture. MSC-seeded mineralized CG scaffolds displayed up-regulation of bone markers osteocalcin and bone sialoprotein as well as depressed expression of chondrogenic markers (collagen II, SOX9, aggrecan) compared to non-mineralized scaffolds. Soluble factor supplementation was shown to further influence cell bioactivity in a dose and cell-type dependent manner with BMP-2 supplementation eliciting significant increases in alkaline phosphatase expression. Efficient (~50%) factor immobilization to the scaffolds via carbodiimide chemistry enabled long-term factor efficacy with minimal effects on scaffold mechanical and microstructural properties. These factors remained bioactive throughout extended (> 7 days) culture periods and elicited similar phenotypic changes compared to equivalent soluble doses, providing a pathway to create biomolecularly patterned scaffolds to drive multi-lineage MSC differentiation.

We describe a CG system where scaffold anisotropy, mineralization, and biomolecule supplementation can be tailored in a spatially-defined manner for TBJ engineering. Ongoing work is integrating mechanical stimulation with biomolecule-immobilized CG scaffolds to more efficiently drive multi-lineage MSC differentiation and long-term phenotypic maintenance across the graded scaffold.

References: 1) Caliari SR et al., Biomaterials, 2011; 2) Harley BA et al., J Biomed Res A, 2010