An In Vitro Model to Test the Influence of Immune Cell Secretome on Mesenchymal Stromal Cell Osteogenic Differentiation

Paree Khokhani, Ruggero Belluomo, Michiel Croes, Debby Gawlitta, Moyo C Kruyt, Harrie Weinans

Published: 1 August 2022


Immune cells and their soluble factors have an important role in the bone healing process. Modulation of the immune response, therefore, offers a potential strategy to enhance bone formation. To investigate the influence of the immune system on osteogenesis, we developed and applied an in vitro model that incorporates both innate and adaptive immune cells. Human peripheral blood mononuclear cells (PBMCs) were isolated and cultured for 24 h and subsequently stimulated with immune-modulatory agents; C-class CpG oligodeoxynucleotide (CpG ODN C), polyinosinic acid-polycytidylic acid [Poly(I:C)], and lipopolysaccharide (LPS); all pathogen recognition receptor agonists, that target Toll-like receptors (TLRs) 9, 3, and 4, respectively. The conditioned medium (CM) obtained from PBMCs after 24 h was used to investigate its effects on the metabolic activity and osteogenic differentiation capacity of human bone marrow-derived mesenchymal stromal cells (MSCs). Conditioned media from unstimulated PBMCs did not affect the metabolic activity and osteogenic differentiation capacity of MSCs. The CM from CpG ODN C and LPS-stimulated PBMCs increased alkaline phosphatase activity (ALP) of MSCs by approximately threefold as compared with the unstimulated control, whereas Poly(I:C) CM did not enhance ALP activity of MSCs. Moreover, direct stimulation of MSCs with the immune-modulatory stimuli did not result in increased ALP. These results demonstrate that soluble factors present in CM from PBMCs stimulated with immune-modulatory factors enhance osteogenesis of MSCs. This in vitro model can serve as a tool in screening immune-modulatory stimulants from a broad variety of immune cells for (indirect) effects on osteogenesis and also to identify soluble factors from multiple immune cell types that may modulate bone healing.

Full Access Link: Tissue engineering. Part C, Methods