The overall burden of osteoarthritis (OA) in the US is estimated at $136.8B annually, an amount that surpasses that of tobacco-related health effects, cancer, and diabetes. Current interventions are not effective in arresting the progressive cartilage degeneration. Stem cell therapy is the new frontier for OA treatment. Intra-articular injection of mesenchymal stem cells (MSCs) derived from different tissue sources has demonstrated its potential to support regeneration of damaged cartilage, thus preventing OA progression. Adipose-derived Stromal Cells (ADSC) are one of the potential candidates for cell therapy. The molecular mechanisms of ADSC-cartilage interactions remain uncertain. We used an ex vivo model of cartilage degeneration to assess metabolism of ADSCs and their effect on diseased cartilage.
ADSC were harvested with the use of disposable closed loop device, Mini-Stem System (Jointechlabs), from donated human lipoaspirates following patients consent. Harvested cell fraction was analyzed by FACS and cell proliferation was assessed by Alamar Blue assay and in culture. Full thickness 4mm cartilage explants from normal ankle joints of 11 human organ donors (9 males and 2 females; age 45-75 yo) were pre-cultured in the presence of IL-1β (1ng/ml or 10ng/ml) to mimic pro-inflammatory environment of the joint. The explants were the incubated with ADSCs encapsulated or not within proprietary hydrogel. The regenerative secretome released by ADSCs was assessed by multiplex ELISA for a representative panel of catabolic and pro-anabolic cytokines. The effect of the secretome on cartilage was evaluated by cell viability, necrosis, and gene expression for cartilage-specific genes. 2 million cells/well were used for culture with cartilage explants. Proteoglycan (PG) content and synthesis in ADSC-cartilage cultures was measured on days 0 (baseline), 3, 10, and 24 and normalized to DNA content using Hoechst 33258 dye. Analysis of pro- and anti-inflammatory mediators released into the culture media was done by Luminex multiplex ELISA of 15 Human cytokines and growth factors.
We found that gel encapsulation provided an adequate support for ADSCs expansion at both high (1 million) and low (250,000) concentrations and that ADSCs survived pro-inflammatory environment. The striking effect of ADSCs on chondrocyte metabolism was identified with PG synthesis. ADSCs alone or embedded in hydrogel induced/elevated PG synthesis above culture control levels (p=0.033 and p=0,036 respectively) and restored PG synthesis inhibited by IL-1β (p=0.058 and p=0.004 respectively). ADSCs also elevated an IL-1RA/IL-1 ratio by 4-fold alone or 7-fold in hydrogels in comparison to IL-1β control (p<0.001).
This study demonstrated that ADSC exhibited 1) a pro-anabolic activity by restoring and stimulating above control levels PG synthesis inhibited by IL-1β and 2) a continuous anti-inflammatory response by upregulating IL-1RA and its ratio to IL-1β. This is the first time when ADSCs were shown to engage in a complex pro and anti-inflammatory paracrine interaction with human adult chondrocytes in vitro. The results of this study suggest that ADSC survive an inflammatory environment, generate anti-inflammatory effect and restore synthesis of cartilage extracellular matrix components. This study supports the further development of the ADSC PoC cell therapy for pre-clinical and clinical trials.