Injectable shear-thinning hydrogels for delivering osteogenic and angiogenic cells and growth factors

Abstract

Bone nonunion may occur when the fracture is unstable, or blood supply is impeded. To provide an effective treatment for the healing of nonunion defects, we introduce an injectable osteogenic hydrogel that can deliver cells and vasculogenic growth factors. We used a silicate-based shear-thinning hydrogel (STH) to engineer an injectable scaffold and incorporated polycaprolactone (PCL) nanoparticles that entrap and release vasculogenic growth factors in a controlled manner. By adjusting the solid composition of gelatin and silicate nanoplatelets in the STH, we defined optimal conditions that enable injection of STHs, which can deliver cells and growth factors. Different types of STHs could be simultaneously injected into 3D constructs through a single extrusion head composed of multiple syringes and needles, while maintaining their engineered structure in a continuous manner. The injected STHs were also capable of filling any irregularly shaped defects in bone. Osteogenic cells and endothelial cells were encapsulated in STHs with and without vasculogenic growth factors, respectively, and when co-cultured, their growth and differentiation were significantly enhanced compared to cells grown in monoculture. This study introduces an initial step of developing a new platform of shape-tunable materials with controlled release of angiogenic growth factors by utilizing PCL nanoparticles.

Fig. 1

A schematic illustration showing a dual-layer structure of STH that can be injected through a customized multineedle injecting device. Osteogenic hydrogels consist of silicate nanoplatelet-based STH that encapsulates osteogenic cells. STH can also incorporate VEGF-loaded PCL nanoparticles to deliver endothelial cells. Injected hydrogel structure can fill the bone defect of any shape and size.

Fig. 2

Biodegradable PCL nanoparticles that can store and release growth factors in STH. a) FESEM images of the synthesized PCL nanoparticles with various particle sizes, which were prepared by different formulation codes (SNP for small-sized nanoparticles, MNP for medium-sized nanoparticles, and LNP for large-sized nanoparticles). b) Cumulative release of the FITC-dextran molecules from the PCL nanoparticles with different diameters of 268±11 nm (SNP, blue triangle), 429 ±17 nm (MNP, red circle) and 743±37 nm (LNP, black square). Fluorescence level of the supernatant of the PCL nanoparticles incubated in PBS was measured by a microplate reader. The error bar represents the standard deviation of the three times experiments. c) The fluorescence microscope image showing a homogeneous dispersion of FITC-dextran encapsulated PCL nanoparticles in silicate-based STH.

Fig. 3

Injectability, storage modulus, shear thinning property, and degradability of PCL nanoparticles-incorporated STH. a) The required injection force of STH (6STH50) to pass through needle was measured using an Instron mechanical tester and 3ml syringe with 18 gauge needle, at a constant rate of 2 mL/min. b) Injection force measurement of PCL nanoparticles-incorporated STH with different preparation formulations was conducted by using an Instron mechanical tester. c) Storage modulus of STHs as a function of nanosilicate amount, solid fraction and PCL nanoparticles. The modulus of elasticity was enhanced by increasing amount of nanosilicate content, solid fraction and the presence of PCL nanoparticles. d) Strain sweep rheology experiments indicated that the linear viscoelastic range decreases with increasing weight percentage of solid weight and PCL nanoparticles in STH. e) Recovery of STHs with and without PCL nanoparticles was monitored by measuring storage moduli and altering high (100%) and low (1%) stain conditions. f) Degradability test of STH in cell culture medium at 37 °C (n=3).

Fig. 4

Photo images of 3D PCL-incorporated STH structures injected by a multineedle injecting system. a) A customized multineedle injecting device that can continuously and simultaneously inject multiple hydrogel types through the single head system. Each hydrogel syringe can be independently actuated by a separate pump. b–d) Stiffer hydrogels to deliver osteogenic cells (7STH50+PCL, blue) and softer hydrogels to deliver endothelial cells (6STH50+PCL, red) could be injected from the 3D injecting device without physical clogging, in both individual (b, c) and simultaneous (d) modes. e) Injected PCL incorporated STHs were sufficiently stable to maintain their dual-layer structures in an aqueous system. f–j) PCL-incorporated STHs could completely fill the complex empty spaces, including triangle, square, and star shaped void structures that were formed in transparent PDMS substrates. k–l) By using a porcine bone, we demonstrated that our injectable bone implant material could fill the defect sites of bone with any size and shape. Panels k and l show photo images of defect site in the porcine bone before and after injecting dual-layer structured STH, respectively.

Fig. 5

Growth and activity of osteogenic cells and vasculogenic cells co-cultured in injected STH material a) Live/dead fluorescent imaging at day 7 of mouse endothelial cells (EC) in monoculture, or co-culture with mouse osteoblasts (EC and OB) with/without the addition of PCL nanoparticles showing live cells in green and dead cells in red. Scale bars represent 100 μm. b) Live cell tracker of EC in red and OB in green cultured for up to 21 days in shear thinning gels with the addition of PCL nanoparticles. Scale bars represent 200 μm. c) Presto blue measurements of monoculture of EC or co-culture with OB, with/without the addition of VEGF-containing PCL nanoparticles to the STH. d) Gene expression within STH with VEGF-containing PCL nanoparticles. Fold change gene expression of co-cultures of BMSCs with HUVECs cells in, relative to monoculture of either BMSCs (for osteogenic genes) or HUVECs (for angiogenic genes). * indicates statistical differences between indicated groups (p<0.05)