Preconditioning of mesenchymal stem cells with mechanobiological load and hypoxia for joint regeneration in moderate osteoarthritis

The presence of osteoarthritic changes in a joint is a contraindication for current tissue engineering approaches, which are designed for regeneration of localized traumatic osteochondral lesions. Our long-term goal is the restoration of degenerative osteochondral lesions and beyond that, the treatment of chondral and osteochondral defects in an OA environment. To be successful we need to understand the constructive and destructive interactions between chondrogenesis, mechanical stress, oxygen tension and inflammation.

 

We propose that mesenchymal stem cells can be preconditioned with mechanobiological load and/or hypoxia prior to implantation in order to achieve a stable chondrogenic phenotype. This preconditioning could allow regeneration of osteochondral defects even in an OA environment.

 

Mechanical load in physiological range is an important factor for maintenance of the healthy status of joint cartilage. On the other hand, overloading is believed to increase the risk of osteoarthritis. Therefore we will apply hydrostatic pressure in different mechanobiological loading regimes to MSCs undergoing chondrogenesis in an in vitro 3D-aggregate culture system. We will identify loading conditions that show the strongest chondrogenic (increased anabolism and decreased catabolism) difference between loaded and unloaded conditions. We plan to achieve this under physiological conditions, but also in OA conditions, which will be mimicked by addition of IL-1ß to the culture medium.

 

Hypoxic culture conditions have also been shown to have beneficial preconditioning for chondrogenic cells. We propose that hypoxia will promote differentiation and suppress markers of hypertrophy in both healthy and osteoarthritic culture conditions. We will apply hypoxia to MSCs undergoing in vitro chondrogenesis and analyse for anabolic and catabolic effects. We will focus on the suppression of hypertrophy in order to achieve a stable chondrogenic phenotype. We plan to identify key signaling pathways involved in the preconditioning process. In particular, we will analyse the PI3K/Akt-dependent pathway, because this cascade is involved in the modulation of chondrogenesis through mechanotransduction and also hypoxia.

Finally we want to examine the effect of MSC-sponge constructs, preconditionend with mechanobiological load and/or hypoxia, to repair posttraumatic osteochondral defects in an animal model in a healthy and an early osteoarthritic condition.

 

Hydrostatic pressure device for stimulation of MSC chondrogenesis

 

 

Mesenchymal stem cell (MSC) chondrogenesis following 21 days differentiation either (a) unloaded or (b) after 7 days loading under hydrostatic pressure during first week of culture. Stained with DMMB for the presence of sulphated glycosaminoglycans

 


 

(a) Phosphotidylinositol-3-kinase (PI3K) as a signalling molecule: Mechanical stimulation or growth factor binding activates the dimerization and phosphorylation of the receptor monomers. A membrane-bound GTP and Ras form a complex, which, in turn, activates PI3K. The resulting effect shows the activation of Akt, also known as protein kinase B. (b) Interactions between different signaling molecules with special focus on mechanotransduction-dependent pathways


Mercator Fellowship of Prof. Dr. Brian Johnstone, Visiting Professor at the Department of Trauma Surgery, University Regensburg Medical Centre

Thematically the Mercator Fellowship is focusing on examination of MSC chondrogenesis under hypoxia and specifically its effect on Hypoxia Inducible Factors (HIFs). Prof. Dr. Johnstone will have three periodical visits during his Mercator fellowship, including an extended three months stay in the lab. He will advise the team on setting up MSC hypoxia culture protocols and validations. Furthermore, he will also visit the other consortium members to develop collaborations within this theme.

 

1st Period of the Mercator Fellowship

 

Prof. Dr. Johnstone visited the laboratory between 16.10.16 – 16.01.17. The exchange involved consulting on various techniques concerning in vitro chondrogenesis, particularly under hypoxic culture, and on the establishment of a rabbit in vivo model of early OA for evaluation of the preconditioned cells. During his visit, he was a keynote speaker and attendee in the ExCarBon meeting at the TIRM Conference (25.11.16-26.11.16 in Regensburg) involving all consortium members, whereby he was involved in discussions regarding the project and collaborations between members that were outlined in the proposal. He also gave two lectures: 1) at the ZMB, entitled “Regeneration potential in an early OA situation from a basic research view”, and 2) at the University Hospital of Regensburg, entitled, “Stem cell tissue engineering: The good, the bad and the ugly”.