Creating 3D Angiogenic Growth Factor Gradients in Fibrous Constructs to Guide Fast Angiogenesis

Abstract

Fast angiogenesis in 3D fibrous constructs that mimic the morphology of the extracellular matrix remains challenging due to limited porosity in the densely packed constructs. We investigated whether mimicking the in vivo chemotaxis microenvironment for native blood vessel formation would stimulate angiogenesis in the fibrous constructs. The chemotaxis microenvironment was created by introducing 3D angiogenic growth factor gradients into the constructs. We have developed a technique that can quickly fabricate (∼40 min) such 3D gradients by simultaneously electrospinning polycaprolactone (PCL) fibers, encapsulating gradient amount of bFGF (stabilized by heparin) into poly(lactide-co-glycolide) (PLGA) microspheres, and electrospraying the microspheres into PCL fibers. Gradient formation was confirmed by fluorescence microscopy. Gradients with different steepnesses were obtained by modulating the initial concentration of the bFGF solution. All of the constructs were able to sustainedly release bioactive bFGF over a 28 day period. The release kinetics was dependent on the bFGF loading and steepness of the gradient. In vitro cell migration study demonstrated that bFGF gradients significantly increased the depth of cell migration. To assess the efficacy of bFGF gradients in inducing angiogenesis, we implanted constructs subcutaneously using mouse model. bFGF gradients significantly promoted cell penetration into the constructs. After 10 days of implantation, a high density of mature blood vessels (positive to both CD31 and α-SMA) were formed in the constructs. Vessel density was increased with the increase in steepness of the bFGF gradient. These gradient constructs may have potential to engineer vascularized tissues for various applications.

Investigation of DNA damage in cells exposed to poly (lactic‐co‐glycolic acid) microspheres

“…To test the genotoxicity of PLGA, 1, 5, or 15 mg of PLGA microspheres were suspended in 0.5 mL media and placed within an insert membrane (pore size 0.4 µm; membrane diameter: 12 mm; nominal pore density: 4 × 10 6 pores per well) that was placed in the well allowing exposure without direct contact. The PLGA doses were selected based on the range of levels previously used for tissue engineering and drug delivery studies . The final concentrations of PLGA microspheres were 0.4, 2, and 6 mg/mL.…”

Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate

“…These methods include internal microfluidic channels [6][7][8], controlled protein release using drug delivery systems [9][10][11][12], gradual immobilization of signalling molecules throughout the scaffold geometry [13][14][15], simple molecular diffusion throughout the scaffold [16,17] or combinations of these methods [18][19][20][21]. The simplest and most representative method is to employ diffusion to generate gradients using a source (So) and a sink (Si) separating a cellladen hydrogel (gel, G) structure (So-G-Si).…”

Computational Model-Based Analysis of Strategies to Enhance Scaffold Vascularization

“…Spatial and temporal gradients of GF affect the direction, structure, rate of cell invasion, and vascularization. 20,21 …”