Matrix deposition on cell-seeded polymeric enthesis repair scaffolds under mechanostimulation

Abstract

Event Abstract Back to Event Matrix deposition on cell-seeded polymeric enthesis repair scaffolds under mechanostimulation Jonathan Tapp1, Mamadou Diallo1, Christopher Alexander1, Joel D. Bumgardner1, Warren Haggard1 and Jessica A. Jennings1 1 University of Memphis, Biomedical Engineering Department, United States Introduction: The incidence of tendon injury is growing; as a result so is the demand for tendon repair strategies[1]. Synthetic polymer fabrics are implemented in tendon repair due to similar mechanical strength properties to a human tendon. The ultimate goal is to improve biocompatibility of artificial tendons. Polymer fabric strips were housed in a custom built bioreactor, seeded with fibroblast and osteoblast cells, and subject to mechanostimulation. The primary objective is to analyze matrix deposition of cell-seeded polymer fabric scaffolds, with and without an applied strain. Methods: The modification of a bioreactor provided controlled, cyclical patterns of strain (Fig 1)[2]. 150x22 mm polyethylene (PET) fabric scaffolds were sterilized by immersion in 2% bleach solution, ultrapure water, and 70% ethanol, followed by thorough rinsing with ultrapure water. The fabric scaffolds were submerged in Dulbecco’s modified eagle media (DMEM) containing 10% fetal bovine serum (FBS) and 100 µg/ml Normocin antibiotic. MC3T3 pre-osteoblast and NIH3T3 fibroblast cells were seeded at a density of 3x 105 cells/mL on separate regions of the scaffold. Once the PET cell scaffolds were secured in the bioreactor, a 5% strain was applied to the region containing fibroblasts for 5 hours daily. The MC3T3 region was kept static. Unstrained scaffolds in petri-dish conditions and bioreactor conditions with media circulation were included as control groups in the 7-day study. Cell DNA growth was quantified via a Picogreen assay, collagen content via a Sirius red collagen detection assay, and glycosaminoglycan (GAG) content via an Alcian blue assay. The amounts of collagen and GAG were normalized using the ratios of matrix component to DNA. Statistical differences between groups were detected using a one-way ANOVA with Holm-Sidak post-test at a confidence level a=0.05. Results: The fibroblast regions of the scaffolds exposed to daily stretching had a higher GAG to DNA ratio than the scaffolds that were only exposed to fluid flow and the control group (Fig 2). Collagen formation was significantly higher on the unstrained scaffolds within the bioreactor with media flow. (Fig 3). Discussion: GAG, a reliable indicator of tendon extracellular matrix (ECM), was deposited in greater amounts when cells were subject to strain. The collagen production of the scaffolds under periodic strain compared to no strain was lower than expected. A possible reason is the static scaffolds were assessed at a point when the cells were in a proliferative phase and producing collagenous matrix at a higher rate. Studies with longer test periods are ongoing to more accurately model the matrix deposition over time, as well as efficacy of cell-seeded ECM-conditioned scaffolds in promoting functional tendon and enthesis repair. Conclusions: The combination of cell seeding with polymer fabrics could lead to improved therapies for tissue repair with minimal inflammation. Parwinder Singh; Jared Cooper; Lauren Eichaker