Student A. B. Lucas Secondary School London, Ontario, Canada
Introduction: In clinics, bone damage is a huge challenge, and often surgical processes such as autographs and allographs have been utilized as treatment. However, insufficient autographs and allographs to meet increasing requests and a chance that plates and screws could fail to hold the grafts in place make this option risky. An alternative would be to utilize hydrogels like Gelatin methacryloyl (GelMA) that do not require grafts. GelMA is a good carrier for drug delivery, has great bio-compatibility, a high consistency with each batch, and has some bone healing properties. ZIF-8, a class of Metal-Organic-Framework (MOF) that contains zinc, is essential for the development and maintenance of healthy tissue. ZIF-8 can be combined with GelMA to both sustain the release of zinc, encourage bone regeneration and strengthen the mechanical properties of GelMA. ZIF-8 will limit the speed at which the GelMA degrades, which helps tissue healing (otherwise the GelMA will degrade before the bone gets a chance to heal), and the porosity of the GelMA, similar to bone marrow, creates a good environment for tissue regeneration and cell growth. In this work, ZIF-8 has been innovatively and successfully integrated into GelMA. The results demonstrate a dramatic increase in calcium deposits in cell cultures compared to pure GelMA, indicating more bone healing.
Materials and
Methods: The creation of ZIF-8 was done by first using 5mL of DI water to solubilize 150mg of zinc nitrate. Second, 300mg of 2-melm was solubilized in 100mL of methanol, and then both resulting solutions were combined and stirred at room temperature for 12 hours. The new solution was then centrifuged at 14, 000 RPM for 30 minutes and consequently washed in PBS, re-suspended in methanol, then centrifuged again 3 times. This is to ensure that there were no impurities. The formation of GelMA was done by preparing gelatin at 10% weight by volume (w/v) in PBS in an Erlenmeyer flask, and it was stirred with a magnetic bar for 1 hour at 60 °C until solubilized. Next, methacrylic anhydride (0.8 mL/g of gelatin) was slowly added dropwise into the stirred solution and subsequently left to stir for another 2 hours at 60 °C. Then, the solution was diluted using 100mL of heated PBS and left to dialyze using a dialysis membrane with water at 50 °C for 1 week. The water was changed 2 times per day to remove unreacted methacrylic anhydride from the GelMA. The GelMA was stored in 50mL Falcons for 4 days at -80 °C and then freeze-dried.
Results, Conclusions, and Discussions: Calcium deposits are an important indicator for bone healing, and cells such as osteoblasts produce more bone by depositing calcium and other materials into any necessary parts of the bone. So, this study completed an Alizarin Red S (ARS) test to compare the osteogenic differentiation between the combination of ZIF-8 and GelMA with pure GelMA by staining calcium deposits for the two. Pure GelMA demonstrated only some calcium deposits because it contains collagen that acts as a scaffold for bone cells, whereas the integration of ZIF-8 and GelMA show significantly more deposits as the zinc helps osteoblasts proliferate, forming new bone, and the GelMA helps the zinc by being a scaffold for the cells. Additionally, zinc in ZIF-8 is essential for the development and maintenance of healthy tissue but will be toxic to cells at a certain point. A calcein AM test was conducted and found that 3mg/mL of ZIF-8 was viable for cells. Next, the degradation of pure GelMA is fast, meaning that the bone cannot heal properly. The addition of ZIF-8 slowed down the rate of degradation and ended up having a 13% increase in weight compared to pure GelMA, helping to sustain drug release and make collagen scaffolds last longer. ZIF-8 increases the toughness of GelMA, leading to a reduction in degradation, aiding in proper bone healing. Next, a qPCR test was done to determine gene expressions and identified OPN and RUNX2. OPN allows for mineralization and the proliferation and adhesion of many bone-related cells. RUNX2 regulates osteoblasts and prevents bone loss. In this study, it was found that the combination possessed more calcium deposits than pure GelMA at 3 mg/mL of ZIF-8, and the qPCR supports evidence of bone healing through identifying OPN and RUNX2 that are important for the proliferation and regulation of bone cells. Lastly, the addition of ZIF-8 to GelMA resulted in a 13% increase in weight compared to pure GelMA, helping the bone to heal. The innovation of combining ZIF-8 and GelMA could be an attractive alternative to treatments like allografts and autografts.
Acknowledgements (Optional): Thank you to Dr. Paul for being my mentor and guide throughout this journey, Choe Choi for aiding me in the process, and my parents for all their unconditional support. I cannot thank all of you enough for all of your help!
