Mesenchymal Stem Cells Delivered in a Novel Cartilage Mimetic Hydrogel for the Treatment of Focal Chondral Lesions in an Equine Animal Model

Abstract

Objectives: A degradable biomaterial has been developed that resembles the native cartilage biochemical properties, in which stem cells can be seeded, differentiate and develop cartilaginous tissue. The purposes of this study were: 1) to determine if mesenchymal stem cells (MSCs) embedded in this cartilage mimetic hydrogel display in vitro chondrogenesis; 2) to demonstrate that the proposed hydrogel can be delivered in situ; and 3) to determine if the hydrogel ± MSCs supports in vivo chondrogenesis. Methods: A photopolymerizable hydrogel consisting of polyethylene glycol, CVPLSLYSGC, chondroitin sulfate (ChS), CRGDS and TGF-β3 was used. Equine bone marrow-derived MSCs were encapsulated in the hydrogel and cultured for 9 weeks. Compressive modulus was evaluated at day 1 and at weeks 3, 6 and 9. Chondrogenic differentiation was investigated via qPCR, Safranin-O staining and immunofluorescence. Three female horses were used. Two 15-mm width x 5-mm depth osteochondral defects were created bilaterally in the medial femoral condyle of each stifle joint. Five groups were established: Hydrogel (n=3), Hydrogel + MSCs (n=3), Microfracture (MFX, n=1), MFX + Hydrogel (n=3), and MFX + Hydrogel + MSCs (n=2). Repair tissue was evaluated at 6 months post intervention with the following cartilage repair scoring systems: macroscopically, International Cartilage Repair Society (ICRS); and histologically, the Modified O’Driscoll scoring (MODS) and ICRS II (Overall assessment 0%, fibrous -100%, hyaline cartilage).The ICRS parameter is scored using a 100-mm VAS, a score of 0 was assigned for properties considered indicative of poor quality and 100 for good quality. Results: In vitro, there was a significant increase in compressive modulus, collagen II and ChS as confirmation of chondrogenesis and hydrogel degradation. (Figure 1) In vivo, the hydrogel was readily photopolimerized in the defect. Cartilage repair was evident in all groups. As shown in Table 1, red indicates best quality score, blue means a poor quality score, but there was no statistical difference. According to the macroscopic ICRS, the hydrogel + MSCs performed better (P= 0.47). However, the MFX + Hydrogel + MSCs tended to perform better per the MODS (P= 0.61); and ICRS-Overall assessment (P= 0.9). Particularly, MFX showed the lowest score for subchondral bone(SCB) abnormalities (0% = abnormal, P= 0.09) but no inflammation was evident (100% = absent, P= 0.53), whereas the Hydrogel had the highest basal integration (100% = complete integration, P= 0.38) but presented moderate inflammation (Figure 2A). MFX showed SCB abnormalities and vascularization (Figure 2 B). Interestingly, a defect treated with MFX + Hydrogel presented more GAGs, less inflammation (vs Hydrogel) and less SCB abnormalities (vs MFX) (Figure 2C). Overall, the group performing better was MFX + Hydrogel + MSCs. Conclusion: This pilot study provides the first evidence of the ability to photopolymerize this novel hydrogel in situ and assess its ability to provide chondrogenic cues for cartilage repair in a large animal model. The presence of all three balanced factors (MFX, Hydrogel, MSCs) had higher scores per MODS summation and ICRS Overall assessment. Strengths of this study include: comparison of standard MFX therapy of osteochondral defects with a novel cartilage mimetic therapy; and use of a large animal that resembles the human knee biomechanically and anatomically. [Figure: see text][Figure: see text][Table: see text]