(497c) Biolithographic Method to Create Spatially Patterned Collagen-GAG Scaffolds with Controlled Microstructure, Mechanics, and Surface Chemistry

The next generation of tissue engineering requires a versatile toolbox of patterned, instructive biomaterials. We have recently developed a suite of technologies that allows us fabricate homologous series of uniform collagen-glycosaminoglycan (CG) scaffold variants with independent control over scaffold microstructural and mechanical properties [1], aligned (anisotropic) CG scaffolds, and multicompartment CG scaffolds that mimic the cartilagenous, osseous, and interfacial regions within osteochondral tissue [2]. Here we describe an approach to generate surface-immobilized patterns and gradients of solution-phase biomolecules onto our CG scaffold systems. This technology enables full, independent control of scaffold microstructure, mechanical properties, and surface chemistry within our CG scaffold system.

CG scaffolds were fabricated from a suspension of type I collagen and chondroitin 6-sulfate via lyophilization [1]. We modified a benzophenone (BP) method recently developed to photochemically pattern 2D substrates to enable BP functionalization of the CG scaffold [3]. An Ar+ Ion laser with photomask was used to expose regions of the scaffold to enable BP-mediated attachment of concanavilin A-biotin (ConA-biotin), Fibronectin (Fn), as well as E- and N-cadherins; patterns were visualized via fluorescent secondary antibodies using an LSM 710 confocal microscope. BP surface density on the CG scaffold surface has been calculated to be greater than 100 per μm². A series of chemical patterns ? stripes (Fig. 1), gradients, multicomponent patterns, bidirectional gradients ? have been created in the CG scaffolds. XRD analysis of the CG scaffold, the BP modified (CG-BP) scaffold, and the Fn functionalized (CG-BP-Fn) scaffold showed small changes in the CG peak post modification, but the scaffold maintains its microstructural properties. There was no observed difference in MC3T3-E1 pre-osteoblast attachment and metabolic activity between CG, CG-BP, and CG-BP-Fn scaffolds at 24 or 48 hours. The Fn ligand has recently been reported to mediate initial cell attachment (< 1 hr) to CG scaffolds [4]. Here we compared MC3T3 attachment to standard CG scaffolds, CG scaffolds that had been soaked in Fn (CG-Fn), CG-BP and CG-BP-Fn scaffolds after 40 minutes; the experiment was performed in PBS to prevent non-specific adsorption of exogenous factors from serum. MC3T3 attachment was significantly higher in the CG-BP-Fn scaffolds compared to all other groups (Fig. 1), suggesting the BP photopatterning method can be used to confer specific bioactivity to CG scaffolds. Ongoing work aims to assess MC3T3 and primary tenocyte bioactivity in response to multiple chemical patterns within the scaffold utilizing differential surface immobilization of Fn, N-cadherin, and PDGF-BB within uniform and anisotropic (aligned) CG scaffold variants.

Fig. 1. (left) 100μm thick stripes patterned through the thickness of a CG scaffold (6mm dia.). (right) MC3T3 attachment is significantly higher in CG-BP-Fn functionalized scaffolds after 30 minutes.

References

1. Harley B, +. (2007) Acta Biomat., 3, 463.

2. Harley B, +. (2010) J.Biomed.Mater.Res.A, 92, 1078.

3. Toh, C, +. (2009) Langmuir, 25, 8894.

4. Sethi, K, + (2002) Wound Repair Regen., 10, 397.