Multiphoton-based micropatterning of bioactive soluble factors : a platform for in vitro reconstitution of soluble cell niche

Abstract

Soluble cell niche components such as growth factors, present either in the interstitial fluid or the extracellular matrix of native tissues, play essential roles in multiple cellular fate processes. Numerous microfabrication and micropatterning technologies have been developed to recapitulate native soluble niche factors in vitro. Nevertheless, quantitatively and spatially controlled micropatterning of such factors remains challenging. Multiphoton-based microfabrication is a promising technology with excellent fabrication resolution, ambient reaction condition and free-form fabrication capability in 2D or 3D configurations. The research team has previously developed a Multiphoton Microfabrication and Micropatterning (MMM) technology to create protein-based microstructures and micropatterns for reconstitution of different cell niche factors, including topological, mechanical and matrix factors, in vitro. However, micropatterning bioactive soluble factors such as growth factors, which can be highly vulnerable to modification, is particularly challenging, and whether the MMM technology can be applied to micropattern them without compromising their bioactivities has not been realized. The present study aims to demonstrate the capability of the MMM technology to micropattern bioactive soluble factors, using Bone Morphogenic Protein 2 (BMP-2) and Wingless-type MMTV Integration Site Family Protein 3A (Wnt3a) as examples, in a quantitatively and spatially controlled manner, and to investigate the bioactivity of the micropatterned growth factors by characterizing the associated signaling events. In a pilot study, BMP-2 failed to trigger Smad signaling after micropatterned by the direct multiphoton-induced cross-linking, suggesting the necessity to develop a better micropatterning method without affecting soluble factors’ bioactivities. A two-step multiphoton-based micropatterning of growth factors has been developed. First, scanning laser-controlled photochemical crosslinking has been used to micropattern NeutrAvidin (NA) on the surface of a microfabricated protein substrate in a quantitatively and spatially controlled manner. Second, BMP-2 has been biotinylated before specific binding to the NA micro-patterns. The bioactivity of micropatterned BMP-2 was characterized by investigating Smad signaling in C2C12 cells. Results showed that the cross-linked NA is functional to bind biotin and the local density of cross-linked NA is controlled by NA concentration, laser power and scan cycle. Moreover, the arbitrary NA micro-patterns can be fabricated according to the region of interest (ROI), suggesting the spatial controllability of NA. Results also showed that the micropatterned biotinylated BMP-2 (bBMP-2) is equally effective as free BMP-2 (fBMP-2) in activating Smad signaling. Moreover, bBMP-2 elicits a more sustained and higher level of Smad signaling than fBMP-2. Finally, activated Smad signaling is only confined to the bBMP-2-bound micro-patterns, suggesting the spatial controllability of BMP-2 soluble niche. Wnt3a was micropatterned via the above two-step multiphoton-based micropatterning method in a pilot study. Results showed that Wnt3a bioactivity is not affected by biotinylation and NA-bound bWnt3a is bioactive to trigger Wnt/β-catenin signaling in L cells. This study develops a two-step multiphoton-based technology to micropattern bioactive soluble factors, using BMP-2 and Wnt3a as examples, in a quantitatively and spatially controlled manner, without compromising their bioactivities. This work further broadens the technical capability of the MMM technology and contributes to in vitro reconstitution of biomimetic soluble cell niche for future applications such as rationalizing for optimal scaffold design.