The Age of hMSC Donor Affects Biomechanical Properties of Tissue Engineered Ligament Constructs
Authors: Novakova Stoyna , Smietana Michael J , Larkin Lisa M 
Published: April 2020

Anterior cruciate ligament (ACL) tears are a major orthopedic challenge. Based on an incidence of 200,000 ACL repairs per year in the United States, the associated cost is in excess of $4 billion annually. Current ACL reconstruction commonly involves the use of allogenic or autogenic tendon grafts. Outcomes for ACL reconstruction with these techniques are limited by graft availability, donor site morbidity, disease transmission, and incomplete integration of native tissue within the surgically created bone tunnel. Thus, there is a demand for alternative tissue sources for ACL reconstruction. Using human derived mesenchymal stem cells (hMSC) as a starting material, we have successfully fabricated scaffold‐less ligament grafts that form their own extracellular matrix and have used these grafts for ACL reconstruction in a sheep model. To better understand the mechanism of graft development during our fabrication process, hMSCs were cultured and harvested at: Day 0 (initial plating), Day 4 (growth phase), Day 8 (differentiation phase), and Day 12 (3‐D construct) for qPCR analysis of bone and ligament markers. At day 15, 3‐D constructs were tested for biomechanics and histology (Fig.
1). The biomechanics data indicated two significantly different cohorts (n>3 per donor per time point) that could be explained by age (young vs. old). In the young cohort (23–25y), the mean tangent modulus of the engineered ligament was significantly stiffer at 19.0 ± 3.2 kPa compared to the old cohort (33–43 y) at 8.7 ± 1.01 kPa. Average gene expression of ligament markers relative to GAPDH were analyzed via qPCR at defined time points during fabrication. We used a combination of gene targets (Collagen 1, Collagen 3, Tenascin C, and Scleraxis) to confirm commitment of the MSCs toward a ligament lineage and to determine any age‐related differences in gene expression. Compared to undifferentiated hMSCs (Day 0), the young cohort showed significantly increased expression of ligament markers Col‐1 & Ten‐C by Day 4, and Col‐3 by Day 8. The old cohort showed an upward trend in the expression of Col‐1 and TenC, but did not reach significance at any time point, compared to age‐matched and undifferentiated hMSCs. Lastly, both the old and young cohort peaked in expression levels of Scleraxis at day 4, followed by a decline (Fig.
2). Additionally, we assessed the presence of collagen in the fully formed constructs with Picrosirius red staining. The images confirm that collagen was present in the constructs at 72 hours following monolayer roll up and there appears to be, qualitatively, less collagen presence in the construct cross sections fabricated from the cells or the 43 y.o donor compared to the constructs fabricated using cells from the 22 y.o donor. In addition, there is an increase in CSA surface area of dense collagen bands in the constructs derived from the cells of young donors compared to the constructs from the older cohort. Based on these data, we conclude that the use of younger donor cells will yield a stiffer and more robust ligament constructs due to the increased generation of ECM. In conclusion, constructs fabricated from the cells of younger donors were stiffer, had collagen deposition, and had earlier expression of ligament markers, all of which will enhance graft effectiveness and clinical outcomes.

Figure 1Figure 2