Subcategory: Biomedical Engineering
Nina Sowah - University of Connecticut
Co-Author(s): Guleid Awale, University of Connecticut, Farmington, CT; Dr. Kevin W.-H Lo, University of Connecticut, Farmington, CT; Dr. Cato T. Laurencin, University of Connecticut, Farmington, CT
The occurrence of bone diseases are increasing rapidly around the world due to factors such as aging population, increased obesity, and poor physical activity. As a result, there is a need for accurate in vitro bone tissue models to understand the biology of bone, analyze bone diseases, and test therapeutic approaches through drug screening (Pirosa 2018). Employing animals, such as rodents, for these purposes can be expensive and requires a great deal of resources. We have developed a bone tissue model to test in the area of drug discovery as a potentially more efficient option. We hypothesize that our model will support osteoblastic differentiation and growth of MC3T3 cells. The process of fabricating the in vitro bone tissue models included making polymeric microspheres, sintering the scaffolds, and constructing the bone tissue model. Testing steps included cell seeding and incubation in media, obtaining scanning electron microscope (SEM) images, and performing matrix mineralization. Porous poly(lactic-co-glycolic acid) (PLGA) microspheres comprised the scaffolds, utilizing phosphate buffered saline (PBS) as a surfactant. The porous inner scaffolds were sintered in 7 mm x 2 mm disc molds at 60°C for 2 hours, and the non-porous outer scaffolds were sintered at 90°C for 90 minutes. The models were constructed by pressing the inner and outer scaffolds together by hand. Prior to seeding, they were sterilized with 70% ethanol and UV light treatments. This study considered the effect of the culture media on cell growth. α-minimum essential medium (α-MEM) served as the control group, and osteogenic medium supplemented with the growth factor bone morphogenic protein-2 (BMP-2) worked as the treatment group. When the incubation periods concluded, the cells were fixed and dehydrated with glutaraldehyde and ethanol/HDMS, respectively. SEM images were obtained to characterize the scaffold morphology and observe cell attachment and spreading onto the model. Matrix mineralization was subsequently performed to quantify late-stage osteogenesis. Our observations showed that the in vitro bone tissue model supported MC3T3 cell growth. Matrix mineralization revealed more mineralized ECM or new bone material on the scaffolds when incubated in osteogenic medium rather than on the scaffolds incubated in α-MEM. The SEM revealed the models to be intact and the cells adhering to the scaffolds. However, there was spacing between the outer ring- and inner disc-shaped scaffolds. Future research endeavors should include exploring methods of increasing PLGA scaffold integrity and further testing to confirm osteogenic differentiation or bone formation. References: Pirosa, Alessandro, et al. “Engineering in-Vitro Stem Cell-Based Vascularized Bone Models for Drug Screening and Predictive Toxicology.” Stem Cell Research & Therapy, vol. 9, no. 1, 2018, doi:10.1186/s13287-018-0847-8
Funder Acknowledgement(s): Funding was provided by the Emerging Frontiers in Research and Innovation - Research Experience and Mentoring program. I thank EFRI-REM, my faculty advisor, and co-authors.
Faculty Advisor: Dr. Cato T. Laurencin, Laurencin@uchc.edu
Role: My role in this research included helping plan the experiments, making the models, conducting the experiments with supervision, and performing analyses.