(3cs) Design and Quantitative Characterization of Spatially-Patterned Collagen Biomaterials for Regenerative Medicine Applications

Regenerating tissue structures such as orthopedic interfaces requires new biomaterial technologies that permit simultaneous control of microstructural, mechanical, and biochemical properties in a spatially-defined manner. My work has focused on the development of spatially-patterned dynamic materials to direct cell proliferation, phenotypic stability, and lineage-specific differentiation with broad applicability to a variety of regenerative medicine challenges.

The first part of my PhD developed and characterized collagen-glycosaminoglycan (CG) scaffolds for tendon tissue engineering. CG scaffolds are regulatory compliant extracellular matrix (ECM) analogs that have been applied to a variety of regenerative medicine challenges. We developed a directional solidification lyophilization approach to fabricate 3D scaffolds with highly aligned pores that mimic the native anisotropy of tendon and other aligned tissues [1]. Contact guidance cues provided by this microstructure support cell alignment within the 3D anisotropic network. We demonstrated that loss of the aligned contact guidance cues led to down-regulation of the tendon marker scleraxis (15-fold), indicating that this structure could aid long-term tenocyte phenotypic stability [2]. In order to improve the mechanical competence of these scaffolds, we created core-shell CG composites inspired by mechanically efficient core-shell composites in nature such as plant stems and porcupine quills that displayed high bioactivity and significantly (~ 36-fold) improved mechanical integrity [3].

We have also developed strategies for integrating multiple biomolecular signals into our scaffolds to provide a platform to simultaneously control multiple cell activities. We found single factor supplementation led to a dose-dependent trade-off between driving tenocyte proliferation versus maintenance of tenocyte phenotype. We then identified supplementation schemes using factor pairs (IGF-1, GDF-5) capable of rescuing tenocyte phenotype and gene expression profiles while simultaneously driving proliferation. We demonstrated a covalent immobilization strategy that allows efficient sequestration of bioactive levels of these factors, making this approach more amenable to in vivo translation. We also developed a benzophenone based direct, photolithographic approach to spatially pattern biomolecules within CG scaffolds and demonstrated creation of a wide range of patterns composed of multiple biomolecular species in a manner independent from scaffold fabrication steps [4].

This work became the foundation to build tendon-bone junction (TBJ) scaffolds with tunable, spatially-patterned microstructural, mechanical, and biochemical properties. TBJ scaffolds were fabricated by combining directional solidification [1] with a previously described liquid-phase co-synthesis method [5] to produce scaffolds displaying a distinct zonal structure with mineral content and geometric anisotropy mimicking the native TBJ. Mesenchymal stem cell (MSC)-seeded mineralized CG scaffolds displayed up-regulation of bone markers osteocalcin and bone sialoprotein as well as depressed expression of chondrogenic markers compared to non-mineralized scaffolds. 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.

I envision my own lab as an international leader in the design and quantitative characterization of 3D materials for applications spanning the interface of engineering and biology. We will leverage the technologies discussed here to design regenerative templates for tissues including bone, cartilage, muscle, and spine. These materials will integrate controlled presentation of therapeutic proteins, drugs, and genes. Additionally, quantitative tools to characterize scaffold permeability and pore size, shape, and fraction should be applicable to porous materials used in catalysis and a wide range of other engineering applications.

References:

1) Caliari SR, Harley BAC Biomaterials, 2011; 32(23): 5330-40.

2) Caliari SR, Weisgerber DW, Ramirez MA, Kelkhoff DO, Harley BAC JMBBM, 2012; in press.

3) Caliari SR, Ramirez MA, Harley BAC Biomaterials, 2011; 32(34): 8990-8.

4) Martin TA*, Caliari SR*, Harley BA, Bailey RC Biomaterials 2011; 32(16): 3949-57.

            *equal contribution

5) Harley BAC, Lynn AK, et al. J Biomed Mater Res A 2010; 92(3): 1078-93.