269411 Immobilization Scheme Utilizing a Photopolymerizable Crosslinker and Biotinylated Fusion Proteins for Neural Tissue Engineering Applications
Recombinant proteins have propelled tissue engineering into new directions through the inclusion of specific moieties that precisely direct cell and tissue behavior. Nerve growth factor (NGF) and semaphorin3A (Sema3A) are two proteins that aid in nervous system development and repair. Implementation of these bioengineered proteins into neural tissue engineering strategies is a novel approach for axon guidance, pruning and regeneration. For these studies, E. coli was transformed with pET-21a+ plasmids incorporating the fusion proteins NGF or Sema3A, and an AviTag sequence, which can be biotinylated using birA enzyme. NGF and Sema3A expression and isolation was successfully preformed using Ni-NTA affinity chromatography and further purified using fast protein liquid chromatography. N-(2-mercaptoethyl)-3-(3-methyl-3H-diazirine-3-yl) propanamide (N-MCEP-diazirine), a heterobifunctional crosslinker, was synthesized for the specific linkage of biotinylated NGF and Sema3A to chitosan films. Biotinylated proteins were also tagged with 5-(and-6)-carboxyfluorescien succinimidyl ester and attached to chitosan films utilizing N-MCEP-diazirine. DRGs were isolated from chicken embryos and placed inside immobilized regions containing either NGF or Sema3A in serum-free growth medium. Controls contained either soluble protein or adsorbed protein directly onto chitosan films. DRGs were imaged under brightfield, and neurite number and extension was quantified with ImageJ software. The amount of protein immobilized to the films compared to adsorbed groups was fluorescently quantified using a spectrophotometer. Our immobilization approach showed that we could specifically control tethering of both proteins at concentrations needed to control cell responses. Engineering and specifically immobilizing these proteins through inserted moieties via a photopolymerizable crosslinker, affords better axon outgrowth utilizing a minimal amount of protein. Our approach for the specific attachment of fusion proteins can be used in a wide variety of tissue regenerative applications. In addition, multiple recombinant proteins can be patterned onto the same substrate through the employment of various protein affinity pairings or bioconjugation techniques.