Forskolin-enhanced microfracture to regenerate hyaline cartilage in chondral defect

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Abstract When critically injured, articular cartilage has an extremely limited capacity for self-healing. Currently, there are several surgical procedures available to treat knee cartilage defects. Given its minimally invasive nature, low cost, and beneficial short-term outcomes, microfracture (MFx), a procedure creating small fractures in the bone, is often considered as the first‐line procedure. However, neotissue in the defect site post-MFx is ultimately remodeled into fibrocartilage, which possesses different biochemical and mechanical properties compared with normal hyaline cartilage, predisposing the repair site to degeneration. Consequently, MFx repairs often fail within several years. Although some MFx procedural modifications have been proposed, evidence regarding their efficacy in humans is lacking. Recently, we found Yes-associated protein (YAP) is highly expressed in the regenerated tissue in the rat knee joint after osteochondral injury, but not in the middle zone of undamaged native cartilage. We then conducted an in vitro screening and found that forskolin, a biosafe herbal compound that stimulates adenylate cyclase activity and indirectly inhibits YAP activation, was able to significantly suppress the transcription and generation of collagen type I (COL1) and alpha-smooth muscle actin (α-SMA) (fibrocartilage markers) , collagen type X (COL10, chondrocyte hypertrophy marker), without compromising the generation of collagen type II (COL2) and glycosaminoglycans (major cartilage components). Therefore, herein we hypothesize that post-MFx forskolin introduction will significantly promote hyaline cartilage formation, thus enhancing the long-term chondral repair outcome after MFx. In this study, we will test the efficacy of two different methods to administer forskolin: repeated injection of forskolin solution (Aim 1), and one-time injection of forskolin preloaded in microparticles for long-term and controlled release (Aim 2). Specifically, in Aim 1, after creating MFx in the knee joint of rats, different doses of forskolin will be injected into the knee joint at weeks 0, 2, and 4 (a total of 3 injections). Eight weeks after surgery, the quality of newly formed tissues will be characterized. In Aim 2, we will generate poly(lactic-co-glycolic acid) (PLGA) microparticles for long-term and controlled release of forskolin, which avoids complications due to repeated injections, such as infection and unnecessary systemic exposure to high dose of drugs. Lastly, several potential MFx-enhancing methods had been previously reported in different publications, but a comparative study to side-by-side assess their efficacy has not been reported. As the first step to address the issue, we will also compare the outcome of the best forskolin treatment conditions, defined from studies in Aims 1&2, to that of a previously published MFx-enhancing technology, specifically the angiogenesis-targeting strategy. By repurposing the biosafe forskolin, the next generation of MFx developed here would provide a minimally invasive and cost-affordable procedure to achieve robust and long-term chondral defect repair, with the neocartilage possessing native tissue cellular phenotype, biochemical composition, and mechanical properties.