Tissue-to-tissue interfaces are commonly present in all tissues exhibiting structural, biological and

chemical gradients serving a wide range of physiological functions. These interfaces are responsible for

mediation of load transfer between two adjacent tissues. They are also important structures in

sustaining the cellular communications to retain tissue’s functional integration and homeostasis. [1] All

cells have the capacity to sense and respond to physical and chemical stimulus and when cultured in

three-dimensional (3D) environments they tend to perform their function better than in two-dimensional

(2D) environments. Spatial and temporal 3D gradient hydrogels better resemble the natural environment

of cells in mimicking their extracellular matrix. [2] In this study we hypothesize that differential functional

properties can be engineered by modulation of macromolecule gradients in a cell seeded threedimensional

hydrogel system. Specifically, differential paracrine secretory profiles can be engineered

using human Bone Marrow Stem Cells (hBMSC’s). Hence, the specific objectives of this study are to:

assemble the macromolecular gradient hydrogels to evaluate the suitablity for hBMSC’s encapsulation

by cellular viability and biofunctionality by assessing the paracrine secretion of hBMSC’s over time. The

gradient hydrogels solutions were prepared by blend of macromolecules in one solution such as

hyaluronic (HA) acid and collagen (Col) at different ratios. The gradient hydrogels were fabricated into

cylindrical silicon moulds with higher ratio solutions assembled at the bottom of the mould and adding

the two solutions consecutively on top of each other. The labelling of the macromolecules was

performed to confirm the gradient through fluorescence microscopy. Additionally, AFM was conducted

to assess the gradient hydrogels stiffness. Gradient hydrogels characterization was performed by HA

and Col degradation assay, degree of crosslinking and stability. hBMSC’s at P3 were encapsulated into

each batch solution at 106 cells/ml solution and gradient hydrogels were produced as previously

described. The hBMSC’s were observed under confocal microscopy to assess viability by Live/Dead®

staining. Cellular behaviour concerning proliferation and matrix deposition was also performed.

Secretory cytokine measurement for pro-inflammatory and angiogenesis factors was carried out using

ELISA. At genomic level, qPCR was carried out. The 3D gradient hydrogels platform made of different

macromolecules showed to be a suitable environment for hBMSC’s. The hBMSC’s gradient hydrogels

supported high cell survival and exhibited biofunctionality. Besides, the 3D gradient hydrogels

demonstrated differentially secretion of pro-inflammatory and angiogenic factors by the encapsulated

hBMSC’s.

References:

1. Mikos, AG. et al., Engineering complex tissues. Tissue Engineering 12,3307, 2006

2. Phillips, JE. et al., Proc Natl Acad Sci USA, 26:12170-5, 2008