<p>The purpose of this research to design a 3D printed tissue engineered nanofiber scaffold with bone marrow mesenchymal stem cells (BMSCs) for the treatment of articular cartilage osteochondral lesions (OCLs) in the pre-arthritic knee. We custom made three instrumental setups and assembled them to produce a three-layered composite scaffold. The scaffold consists of UV light-cured polyethylene glycol diacrylate (PEGDA) hydrogel layer as cartilage, 3D printed polycaprolactone (PCL) filament cross-linked architecture as subchondral bone layers and an electrospun PCL nanofiber layer covering the scaffold all around. The goal is to conduct in vitro biological analysis of rabbit BMSCs seeded PEGDA-PCL samples. The objectives of the RCSA project are (1) analysis of cell viability, adhesion and proliferation of cell-seeded PCL-PEGDA scaffolds. (2) study of cell attachment on the PCL-PEGDA scaffolds and their morphological distribution by scanning electron microscope (SEM).(3) histological study of the cell-scaffold constructs by flash freezing method. This study used passage 6 (P6) Stem Pro™ BM mesenchymal stem cells for the cell viability, cell adhesion, and proliferation assays. Cells was cultured (35,000 cells/ mL of cell culture medium) for 3, 7, and 14 days under standard cell culture conditions. The Invitrogen™ AlamarBlue™ Cell Viability Reagent (ThermoFisher Scientific # DAL1025) was used to determine the number of viable cells on each of our scaffolds. The absorbance of each scaffold will be measured using a spectrophotometer set at 570nm. Rabbit BMSCs attached to scaffolds were visualized using SEM. After 1,7,14 and 21 days, cell seeded onto scaffolds was fixed in 4% formaldehyde for 30 minutes at room temperature. SEM images was performed after dehydrating the scaffolds using ethanol. The cell – scaffold (PEGDA-PCL sample) constructs cultured in rabbit growth medium was assessed for histological analysis. Samples was fixed with 4% paraformaldehyde (PFA) and incubated at room temperature for 48 h. Then each sample was transferred into 30% sucrose in 1×PBS at 4°C until each sample sinks. After embedding within optimal cutting temperature (O.C.T.) medium, each scaffold was flash freeze by pouring liquid nitrogen (N2). Then serial cryosections was done and each section was stained with Safranin-O (for collagen). Image was taken by bright-field or polarized light microscopy. This study found statistically significant cell viability difference (optical density 570 nm) between without and with BMSC cell seeded PCL-PEGDA PEGDA for 3 and 7 days. SEM analysis indicated BMSC cell attachment on the PCL nanofiber layer, but no presence of cells at the subchondral bone layers at 14 days of culture. Histology analysis on cell seeded scaffold is currently underway and confocal microscopy analysis data will be reported in the draft manuscript. Based on available data, we can conclude that our PCL-PEGDA can be used as a foundation for cartilage scaffold.</p>

In Vitro Evaluation of Pegda-Pcl Scaffold for Cartilage Regeneration

Paper Proceedings: https://asmedigitalcollection.asme.org/IMECE/IMECE2021/volume/85543

ASME 2021 International Mechanical Engineering Congress and Exposition (IMECE2021)

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