The role of the bone marrow niche and G-CSF in osteochondral repair

Osteoarthritis is a debilitating condition with an increasing global burden. Regenerative strategies to treat damaged articular cartilage aim to relieve pain and maintain joint function. Procedures for osteochondral repair, such as drilling or microfracture of the subchondral bone, are thought to restore cartilage by delivering bone marrow cells to the defect site. However, the specific identity or contribution of these cells to the repair process is not well understood. Mesenchymal stem/stromal cells are thought to contribute to tissue repair by either progenitor or trophic action. In the bone marrow niche mesenchymal stem/stromal cells (BMSCs) contribute to haematopoietic stem and progenitor cell (HSPC) maintenance and the dynamics of this are disrupted by osteochondral repair procedures. In addition, the bone marrow niche can be further manipulated by the HSPC mobilising agent granulocyte-colony stimulating factor (G-CSF). This cytokine has previously been shown to improve the repair of osteochondral tissues, however, the cellular mechanisms underlying this response are poorly described. This thesis investigates BMSC and HSPC interactions in bone and cartilage repair, and the mechanism by which G-CSF may act to improve repair outcomes. To look at BMSC–HSPC interactions a co-culture system was designed and then used to demonstrate that HSPCs inhibit the differentiation of BMSCs towards osteogenic and chondrogenic lineages. To explore the interaction and role of these cells in vivo during osteochondral repair, a mouse defect model was established. Defects were 729 (± 42.6) µm wide at the articular surface, occupied 41.6 (± 2.5) % of the width of the patella groove and penetrated 1205 (± 58.6) µm into the distal femur. The coefficient of variation (CV) for each parameter was below 6%. The area of injury demonstrated regenerated articular cartilage, an observation not previously described in the typically poor healing C57Bl/6 strain of mice. Further, cells known to respond to G-CSF were seen to have specific spatial profiles in the osteochondral repair tissue over time. For example at 24 hours post-surgery, defect sites were filled with a blood clot within which neutrophils (anti NIMP) were embedded. After 1 week, these cells were no longer present within the injury whilst macrophages (anti-CD68) had infiltrated the wound site having previously been rarely observed at 24hrs. To begin to understand the role of BMSCs in this repair model, Nestin-GFP reporter mice were studied. GFP signal was detected at the defect site at 24 hours after injury. Mice treated with G-CSF showed an increase in Nestin-driven GFP in their spleens. In addition, GFP was detected in the defect of G-CSF treated mice at 4 days post-surgery. To explore the role of G-CSF on BMSCs directly, in vitro techniques were used. These studies demonstrated the presence of a functional G-CSF receptor in the non-haematopoietic bone marrow-derived BMSCs. The impact on MSC cell function was explored, including proliferation and differentiation, showing minimal impact and suggesting a more likely role for G-CSF in the regulation of the trophic activity of BMSCs. The data presented in this thesis further illustrates that the cross talk of multiple bone marrow cell types influences their cell fate. The osteochondral model developed during these studies can be exploited further to investigate the role of individual cell types during repair. In addition, the studies presented here illustrate that the actions of G-CSF are likely exerted on a diverse range of cell types during the repair process and a better understanding of the mechanism of action will allow greater precision, translation and clinical utility.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2019

Author dc:creator

• Beaton, Francesca Emily

Advisors dc:contributor.advisor

• McCaskie, Andrew
• Birch, Mark

• Bone
• Cartilage
• Mesenchymal
• Wound repair
• Granulocyte
• Niche
• Osteoarthritis
• Microfracture
• G-CSF
• Osteochondral

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/90d524d7-e913-4ad8-a3dc-52c8389d08e9/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.39459

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/292309