Proteoglycan-4 (PRG4) is a mucinous glycoprotein with critical roles in the bio-lubrication of articular cartilage and joint health1. Intra-articular injection of PRG4 protects against cartilage deterioration in in vivo models of osteoarthritis, an effect that is largely attributed to restored bio-lubrication. Thus far, tissue-engineered, collagen-glycosaminoglycan (coll-GAG) scaffolds2 developed by our lab have shown significant promise in treating focal yet critical-sized osteochondral defects within several in vivo animal models and in humans Such scaffolds alone, however, lack the requisite frictional qualities of articular cartilage. The regenerative capacity of collagen-based biomaterials can be improved through the incorporation of therapeutic molecules4 and we have previously demonstrated that the frictional properties of coll-GAG scaffolds are enhanced by soaking scaffolds in recombinant human (rh)PRG4-solution in vitro3. The goal of this study was, therefore, to investigate an alternative approach to PRG4-injections by developing a scaffold-based recombinant human (rh)PRG4 delivery system for superior bio-lubrication of cartilage biomaterials.
1) The effect of rhPRG4 (Lµbris Biopharma3) treatment on mesenchymal stem cell (MSC) proliferation (DNA, Picogreen) and sulfated-GAG (sGAG, Blyscan Assay) production was examined in 2D-culture (wells). 2) Subsequently, rhPRG4 was incorporated via bulk-addition to a collagen I-hyaluronan slurry (blended mixture) and lyophilized, previously optimized for controlled molecule release4. The coll-rhPRG4 scaffold properties were assessed including pore architecture (JB4, toluidine-stain), rhPRG4 release (ELISA) and molecular weight (western blot). 3) The scaffolds’ mechanical properties including the bio-lubrication (coefficient of friction3, COF) and compressive stiffness were characterized. 4) Finally, the biological response of MSCs in 3D-culture (scaffolds) after 14, 21, and 28 days was assessed.
1) In 2D, rhPRG4-treatment significantly increased MSC proliferation (+61%) and sGAG production (+4.3x). 2) rhPRG4 was successfully incorporated within scaffolds as demonstrated by western blots indicating intact rhPRG4 of molecular weight ~460kDa, rhPRG4 released gradually from the scaffold up to day 14 (67% release by day 7). The novel biomaterial was highly porous with uniformly interconnected pores (121.9 to 160µm). 3) The coll-rhPRG4 scaffolds were lubricious, with a 42% reduction of COF (Coll-GAG-rhPRG4: 0.068 ±0.01 vs. Coll-GAG: 0.118±0.02, p<0.001), however incorporation of PRG4 reduced the stiffness of the scaffolds. 4) Coll-GAG-rhPRG4 scaffolds produced 40 to 80% more sGAG than controls across multiple timepoints (Days 14, 21, and 28).
An innovative scaffold based rhPRG4 release system was successfully designed for cartilage repair applications. Additionally, beyond rhPRG4’s ability to support bio-lubrication, a previously undescribed biological effect was revealed, rhPRG4 increased cell proliferation and sGAG formation in vitro. Therefore, PRG4 containing biomaterials may be suitable for other applications (e.g., contact lens materials). To conclude, scaffold-based delivery of PRG4 may circumvent the need for multiple intra-articular injections whilst also providing cues to promote cartilage regeneration.
1. Jay et al, Matrix Biology, 2014; 2. Levingstone et al, Biomaterials, 2016; 3. Matheson et al, Journal of the Mechanical Behavior of Biomedical Materials, 2021; 4. Almeida et al, Acta Biomaterialoa, 2014.
ReCaP: European Research Council Advanced Grant (Grant: 788753), National Science Foundation- Science Foundation Ireland (NSF-SFI) (Grant: NSF_ 17_US_3437), ADMIRE Marie Sklodowska-Curie-Action-Cofund (EU Horizon 2020: 945168, SFI: 12/RC/2278_2)"