Mesenchymal stem cells (MSCs) have the ability to differentiate into a wide range of specialized cell types, such as adipocytes, chondrocytes, and osteoblasts. MSCs have the potential use for tissue regeneration in three-dimensional (3D) scaffold through the control and guidance of MSCs differentiation. However, as there is little understanding of mechanisms for MSCs differentiation in biomaterials, it is still difficult for the regeneration of viable complex three-dimensional (3D) tissues from constructs of stem cells and biomaterials. The biomolecules and their concentrations in biomaterials have important impacts on MSCs behaviour. Therefore, it is of particular interest to fabricate a supportive three-dimensional (3D) biofunctional biomaterial with spatial control of biomolecules and their concentration as a platform to more effectively study MSCs adhesion, proliferation and differentiation with biomaterials.

In this study, a microfluidic gradient generator was developed as a platform to fabricate poly(ethylene glycol) (PEG) hydrogel with gradient distribution of arginine-glycine-aspartic (RGD) peptide. The effect of RGD and its concentration on MSCs differentiation was studied. The gradient PEG hydrogel can achieve identity and concentration control of biomolecules. RGD peptide is a biomolecule and often used for enhancing cell adhesion. Moreover, it was also found to have impacts on stem cells differentiation. Therefore, the effect of RGD peptide on MSCs differentiation was studied.

In order to fabricate RGD gradient PEG hydrogel, a PDMS microfluidic gradient generator was designed and fabricated using photolithography and soft lithography technique. Simulations were done to find the optimal parameters to achieve stable and continuous bio-molecule gradient. After fabrication of the PDMS microfluidic gradient generator, flow characterization was explored to find the optimal flow parameters for the generation of colour gradient of dye solution.

RGD peptide was incorporated to PEG molecule to form acrylate-PEG-RGD (ACRL-PEG-RGD) by the reaction of -NH2 group of RGD with -NHS group of ACRL-PEG-NHS. Fourier-transform infrared spectrometer (FTIR) was used to characterize the conjugation reaction. The RGD gradient PEG solution was then formed using the PDMS microfluidic gradient generator by injecting the PEG-DA solution with/ without ACRL-PEG-RGD into the two inlets. The RGD gradient PEG hydrogel was then solidified after UV polymerization.

MSCs were then cultured on two dimensional (2D) RGD gradient PEG hydrogel. With the increase of RGD concentration on the gradient PEG hydrogel, the adherent cell density and single cell spreading area increased. The impact of different RGD gradients on MSCs adhesion was also studied. It was found that there was a critical concentration, below which fewer cells can attach on the hydrogel surface. The effect of RGD gradient on MSCs orientation or alignment was also explored.

MSCs were encapsulated into PEG hydrogel with different RGD concentrations for three dimensional (3D) cell culture. The cells viability was tested by live/dead assay. MSCs were induced to osteogenic differentiation. Vonkossa staining of mineralization was used to characterize the osteogenisis. The results showed that the cells viability increased with the increase of RGD concentration. RGD peptide can promote the osteogenisis of MSCs in osteogenic medium. MSCs were also encapsulated into PEG hydrogel with RGD concentration gradient with UV polymerization. The stem cells encapsulated in the RGD gradient PEG hydrogel were induced to osteogenic differentiation. The effect of RGD gradient on cells viability and osteogenisis was studied.