Abstract
Introduction and Objective
Mesenchymal stem cells (MSC) are attractive candidates for bone regeneration approaches. Benefits of MSC therapy are mainly attributed to paracrine effects via soluble factors, exerting both immunoregulatory and regenerative actions. Encapsulation of MSC in hydrogels prepared with extracellular matrix (ECM) proteins has been proposed as a strategy to enhance their survival and potentiate their function after implantation. Functional activity of MSC can be regulated by the physical and mechanical properties of their microenvironment. In this work, we investigated whether matrix stiffness can modulate the crosstalk between MSC encapsulated in collagen hydrogels with macrophages and osteoblasts.
Materials and Method
Collagen hydrogels with a final collagen concentration of 1.5, 3 and 6 mg/mL loaded with human MSC were prepared. Viscoelastic properties of hydrogels were measured in a controlled stress rheometer. Cell distribution into the hydrogels was examined using confocal microscopy and the levels of the immunomodulatory factors interleukin-6 (IL-6) and prostaglandin E2 (PGE2) released by MSC were quantified by immunoassays. To determine the effect of matrix stiffness on the immunomodulatory potential of MSC, human macrophages obtained from healthy blood were cultured in media conditioned by MSC in hydrogels. The involvement of IL-6 and PGE2 in MSC-mediated immunomodulation was investigated employing neutralizing antibodies. Finally, the influence of soluble factors released by MSC in hydrogels on bone-forming cells was studied using osteoblasts obtained from trabecular bone explants from patients with osteonecrosis of the femoral head during total hip arthroplasty.
Results
MSC loaded in hydrogels containing varying concentrations (1.5, 3 and 6 mg/mL) of collagen were viable. Rheology measurements determined that the hydrogel stiffness increased with increasing collagen concentration. Encapsulation of MSC into hydrogels barely affected their storage modulus values. MSC acquired a three-dimensional (3D) arrangement in all hydrogels and showed a more elongated shape in hydrogels with higher stiffness. The secretion of IL-6 and PGE2 by MSC in hydrogels increased with increasing matrix stiffness. Media conditioned by MSC encapsulated in stiffer hydrogels decreased TNF-α levels secreted by macrophages to a higher extent than media conditioned by MSC in softer hydrogels. This effect was partially mediated by PGE2. Finally, our preliminary results indicated that factors released by MSC in hydrogels regulated osteoblast-mediated mineralisation and this effect was dependent on hydrogel stiffness.
Conclusions
Our data indicate that matrix stiffness of collagen hydrogels regulates the production of soluble factors by MSC and their paracrine actions on macrophages and osteoblasts.
