Tensile Loading Promotes Fibrochondrocyte Differentiation of Mesenchymal Progenitors and Initial Development of Engineered Fibrocartilage Constructs

Introduction: Bone marrow stromal cells (BMSCs) are an attractive cell source for fibrocartilage repair due to their multipotency and availability. Previous studies have shown that transforming growth factor β1 (TGF-β1) and dexamethasone stimulate the chondrogenesis of BMSCs in 3D culture[1], but the types or combinations of signals required to drive fibrochondrocyte differentiation remain unclear. Given the significant levels of tensile stresses experienced by fibrocartilage tissues [2], tension may play an important role in fibrocartilage development. The goal of this study was to examine the potential for cyclic tensile loading to promote fibrochondrocytic differentiation of BMSCs and the production of a fibrocartilage-like tissue construct. Materials and Methods: Bovine BMSCs were isolated from the tibiae and femora of an immature calf and expanded in the presence of 10% FBS and 1ng/ml basic-FGF. Third passage BMSCs were seeded into fibrin gels at 10e6 cells/ml and cultured in chondrogenic medium (DMEM, antibiotic/antimycotic, ascorbate, Lproline, ITS, NEAA, aprotinin, 10ng/ml TGF-β1, and 100nM dexamethasone). After 7 days of pre-culture, the constructs were either loaded under cyclic tension (10% peak-peak displacement at 1Hz, 6 x 1h on/3h off each day), held at 0% displacement as static controls, or maintained in free-swelling/contracting (FS) conditions. After 7 (day 14) or 14 (day 21) days of loading, digested constructs were analyzed for sGAG, total collagen, and DNA contents and media samples were assayed for released sGAG. Gene expression was measured by real-time RT-PCR, and proteoglycan processing was examined via western blot analysis and immunofluorescence imaging. Data were analyzed by general linear models and are presented as the mean ±SEM. Results: During the 7 day pre-culture, BMSCs contracted the fibrin matrix by 30% from 20mm to an average length of 13.9±0.35mm, and cells continued to contract the FS gels to 9.68±0.20mm and 8.75±0.13mm after 14 and 21 days, respectively. By day 21, this contraction resulted in a 37.5% difference in construct length for the 0% and +10% groups compared to FS. Construct DNA contents did not significantly vary during the 2 week loading period (not shown). By day 14, cyclic tension significantly increased the total construct sGAG content above the FS (12.5% higher) and 0% (10.6% higher) conditions (Fig 1A) and significantly increased the collagen content over FS (33.7% higher) and 0% (19.0% higher) conditions (Fig 1B). No significant differences in sGAG or collagen contents were detected at day 21. Cyclic tension significantly increased collagen I mRNA expression over the FS group at days 14 and 21, but had no effect on collagen II, aggrecan, or osteocalcin expression (not shown). Full length G1-reactive aggrecan (450kDa) was detected by western blot analysis of construct extracts, and the amount of aggrecan within the constructs appeared to increase with time in culture (Fig 2A). The primary G1-reactive form of processed aggrecan within the constructs was approximately 70kDa, characteristic of aggrecanase (ADAMTS-4/5) mediated cleavage [3]. Native fibrocartilage (FC) contained higher levels of degraded aggrecan than articular cartilage (AC). The 0% and +10% loading groups released significantly more sGAG to the media (Fig 1C) than the FS group. Two major forms of G3-reactive aggrecan were detected in the conditioned media at approximately 55kDa and 140kDa (Fig 2B), with higher levels in the day 20 media of the 0% and +10% groups than in the FS group. Consistently, less G3 immunofluorescence staining was also observed in sections from the 0% and +10% constructs than in those from the FS group (Fig 2C). Discussion: Overall, the results of this study provide preliminary evidence that combinations of chondrogenic biochemical stimuli and tensile stresses have the potential to promote a fibrochondrocyte-like phenotype in differentiating BMSCs and to stimulate the development of a fibrocartilage-like matrix. Cyclic tensile loading specifically stimulated collagen I mRNA expression and promoted increases in total collagen accumulation during the first week of loading. However, the similar levels of matrix accumulation in all groups by day 21 suggest that BMSCs became less responsive to this cyclic tension protocol with extended loading periods, perhaps due to changes in matrix structure or altered cellular sensitivity. Therefore, the specific loading and culture conditions may need to be optimized for sustained stimulation of matrix synthesis. Cyclic tension also stimulated sGAG production, and the tensile stresses generated by cellular contraction in the constrained gels (0% and +10%) appeared to increase aggrecan catabolism. The higher levels of processed aggrecan in native FC compared to AC suggest that increased aggrecan catabolism is also a marker of the fibrochondrocyte phenotype and provide additional evidence that tensile stresses promote fibrochondrocyte-like differentiation of BMSCs. References: [1] Caterson et al. Biomed Mater Res 57: 394-403, 2001. [2] Messner and Gao. J Anat 193: 161-78, 1998. [3] Sandy et al. Biochem J 335: 5966, 1998. Acknowledgements: NSF GTEC ERC (EEC 9731643)