Nanofibrous scaffold with incorporated protein gradient for directing neurite outgrowth

Abstract

Concentration gradient of diffusible bioactive chemicals assumes many important roles in regulating cellular behavior. Among the many factors influencing functional recovery after nerve injury, such as topographical and biochemical signals, concentration gradients of neurotrophic factors provide chemotactic cues for neurite outgrowth and targeted renervation. In this study, a concentration gradient of nerve growth factor (NGF, 0-250 μg/ml) was incorporated throughout the thickness of poly(ε-caprolactone)-poly(ethylene glycol) coaxial electrospun nanofibrous scaffolds (∼700 μm thick with ∼800 nm average fiber diameter). The existence of the protein gradient upon protein release was demonstrated using a customized under-agarose-PC12 neurite outgrowth assay. When exposed to scaffolds endowed with NGF concentration gradient (NGF-CG), a significant difference in the percentage of cells bearing neurite outgrowth was observed (7.1 ± 1.9% vs. 0.8 ± 0.3% for cells exposed to high vs. low concentration surface, respectively; p < 0.05). In contrast, no significant difference was observed when cells were exposed to scaffolds that encapsulated a fixed concentration of NGF. Direct culture of PC12 cells on the substrates demonstrated the cytocompatibility and the effect of diffusible NGF gradient on neurite outgrowth. A significant difference in the percentage of cells with neurite extensions was observed when PC12 cells were seeded on NGF-CG scaffolds (21.2 ± 3.6% vs. 10.4 ± 1.3% on high vs. low concentration surface, respectively; p < 0.05). Furthermore, Z-stack confocal microscopy tracking of neurite extensions revealed the chemotatic guidance effect of NGF concentration gradient. Directed and enhanced neurite penetration into the scaffolds towards increasing NGF concentration was observed. In vitro release study indicated that the encapsulated NGF was released in a sustained manner for at least 30 days (80.4 ± 3.6% released). Taken together, this study demonstrates the feasibility of incorporating concentration gradient of diffusible bioactive chemicals in nanofibrous scaffolds via the coaxial electrospinning technique.

Three-dimensional aligned nanofibers-hydrogel scaffold for controlled non-viral drug/gene delivery to direct axon regeneration in spinal cord injury treatment

“…More importantly, our scaffold design allows robust drug/gene encapsulation. Our previous works have shown that electrospinning enabled the incorporation of a wide variety of drugs ranging from proteins to low molecular weight lipophilic drugs and nucleic acids within nanofiber matrices 16 17 18 19 20 21 22 23 24 25 26 . Collagen hydrogel, on the other hand, is well-established in its ability to enable sustained drug/gene delivery 27 28 29 .…”

Microfiber drug/gene delivery platform for study of myelination

“…Moreover, we have previously employed similar electrospun substrates for the delivery of proteins and low molecular weight drugs [27][28][29]. Thus, this microfiber substrate could be a versatile functional drug/gene screening platform for OLs.…”

Concentration Gradients in Material Sciences: Methods to Design and Biomedical Applications

“…designed poly(ε‐caprolactone)‐poly(ethylene glycol) coaxial electrospun nanofibrous scaffolds in which a gradient of NGF was incorporated (0–250 µg mL −1 ). [ 67 ] When cells were in contact with scaffolds containing the gradient, a higher percentage of cells bearing neurite outgrowth was observed, when compared with scaffolds containing a fixed NGF concentration. [ 67 ] Also, Chang et al.…”

“…[ 67 ] When cells were in contact with scaffolds containing the gradient, a higher percentage of cells bearing neurite outgrowth was observed, when compared with scaffolds containing a fixed NGF concentration. [ 67 ] Also, Chang et al. designed a gelatin‐based nerve conduit, mimicking the architecture of a nerve, with spatial gradients of neurotrophic factors (NGF and brain‐derived neurotrophic factor (BDNF)) either encapsulated in gelatin nanoparticles (for controlled release) or directly incorporated in the gelatin‐scaffold.…”