Characterisation of Multipotential Stromal Cells (MSCs) from Subchondral Bone in Ankle Osteoarthritis and their Biological Modulation Towards Chondrogenesis Enhancement

Osteoarthritis (OA) of the ankle is unique in that it has an early onset and is often post-traumatic. Surgical treatments of ankle OA are less effective than in other joints due to its anatomical and biomechanical complexity, whereas joint-preserving treatments, which rely on endogenous multipotential stromal cells (MSCs), show good outcomes of up to 2 years, but degenerate subsequently. The aims of this study are: 1) To develop understanding of OA progression in the talus and tibia of the ankle through micro computed tomography, histology with cartilage damage grade scoring (Osteoarthritis Research Society International cartilage histopathology assessment (OARSI)) and immunohistochemistry, with a focus on resident reparative cells, CD271+ MSCs. 2) Assess the presence and potency of resident MSCs in OA talus and tibia in comparison to non-diseased, iliac crest (IC) MSCs, utilising standard in vitro methods 3) Develop two-dimensional expansion methods of MSC preconditioning for chondrogenesis enhancement, using clinically relevant, xeno-free biological stimulants, Kartogenin (K) and Platelet Lysate (PL) 4) Investigate preconditioning and 3D culture for synergetic enhancement of MSC chondrogenic differentiation in non-diseased IC MSCs, and OA talar and tibial MSCs, utilising fibrin alginate and pellet culture methods for GAG deposition and gene expression. In non-diseased bone, the tibia presented a significant, 2.5-fold higher bone volume of total volume (p=0.001) than the talus. In OA, the subchondral bone plate of talus and tibia thickened 1.3-to-2.5-fold compared to non-diseased controls, with bone volume of total volume increasing within 2 mm from the subchondral bone plate, but falling below non-diseased controls after 2 mm in OA in both talus and tibia. The subchondral bone plate thickened 3-7 fold under damaged cartilage (OARSI grade 5) cartilage compared to grade 3 in both talus and tibia (p=0.003, 0.0003 respectively), and bone volume of total volume increased 2-fold in tibia under grade 5 compared to grade 3 cartilage (p=0.002), but not in talus. MSCs appeared to associate with cartilage damage, with CD271+ positive staining correlating significantly with overlying cartilage damage grade in tibia (p=0.008 between grade 3 and 6), with a trend also clear in the talus. CD271+ MSCs were also found in similar areas as CD56+ osteoprogenitors and E11+ osteocytes, suggesting a link between MSCs and new bone formation. Plastic adherent, colony-forming MSCs were found in both OA tibia and talus, with a significant 30-fold higher MSC proportion of total cells than IC (p=0.005, 0.013 respectively). OA talar and tibial MSCs showed 1.5-3-fold increased osteogenesis by calcium deposition, and 2-fold increased chondrogenesis by GAG production, significant in the talus (p=0.012). Talar MSCs showed a 10-fold reduction in adipogenesis compared to IC and tibial MSCs by Oil Red O staining normalised to DAPI (p=0.049). Preconditioning with xeno-free 10 %human serum (HS) and biological stimulants 10 μM K and 10% PL showed increased proliferation in IC MSCs compared to the positive control ChondroDIFF. Treatment with K led to increased expression of chondrogenic gene ACAN, but reduced COMP, COL2 and SOX9 compared to ChondroDIFF, whereas PL did not elevate expression of any chondrogenic gene. Subsequent differentiation in 3D culture showed a trend for elevated GAG production and retention after preconditioning with K/10% HS, and differentiation in K/50% HS relative to other conditions, suggesting this as the most successful combination for cartilage regeneration. PL treatment alone led to the majority of GAG not being retained, and elevated expression of osteogenic genes, as such appears unsuited for chondrogenic preconditioning of OA talar and tibial MSCs. Ankle OA cultures showed reduced GAG retention in comparison to IC, and far more variance in GAG production, suggesting OA has an impact on the chondrogenic ability of MSCs following biological stimulation. Overall, OA induced profound changes in talar and tibial cartilage and bone structure, likely impacting on joint biomechanics. MSCs are present within bone and are consistent with therapies which rely on endogenous cells, e.g. microfracture, however their association with bone-forming cells indicated a predominantly osteogenic phenotype. In standard conditions, these MSCs are capable of chondrogenesis to a greater extent than IC, but require stimulants which block osteogenesis whilst enhancing chondrogenesis to be targeted clinically. Preconditioning with K/10% HS demonstrated the ability to initiate chondrogenesis, and subsequent differentiation in K/50% PL shows enhanced GAG production and reduced RUNX2 expression, making this a suitable candidate for a clinically suitable drug to enhance chondrogenesis in combination with microfracture.