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Tissue Engineering

The effects of Dexamethasone and BMP-2 delivered at specific circadian time points on bone regeneration

(I-321) The effects of Dexamethasone and BMP-2 delivered at specific circadian time points on bone regeneration

Thursday, October 12, 2023

2:00 PM – 3:00 PM PDT

Location: Exhibit Hall - Row I - Poster # 321

Presenting Author(s)

SM

Sophia M. Millan (she/her/hers)

Undergraduate Research Assistant
University of Massachusetts Amherst
Amherst, Massachusetts, United States

Co-Author(s)

DM

Dorcas M. Matuwana, n/a

PhD Student
University of Massachusetts Amherst
Sunderland, Massachusetts, United States

Primary Investigator(s)

CK

Cathal Kearney

Assistant Professor
University of Massachusetts Amherst, United States

Introduction::

Osteoporosis diseases are rampant in the US with 10 million people suffering from it. This disease leads to fractures and multiple complications caused by impaired osteogenesis negatively impacting bone strength. Dysregulated circadian rhythms (CRs) is one cause of osteoporosis. CRs are a 24 hour cycle, responsible for coordinating physiological and behavioral processes that can be entrained by stimuli. In vitro, we can synchronize circadian rhythms by using temperature, or glucocorticoids. Here, MC3T3 pre-osteoblasts will be used to investigate bone regeneration in in vitro assays. Previous studies using this cell line have shown glucocorticoids at low doses can reset circadian rhythms, and that bone morphogenetic proteins have a positive effect on bone formation. Dexamethasone, (a glucocorticoid) plays an important role during osteoblastic differentiation. Bone morphogenetic protein-2 (BMP-2) is a growth factor that promotes osteoblast maturation. Hence, dexamethasone and bone morphogenetic protein-2 were chosen for this project for their specific roles in osteogenesis. While independent delivery of these treatments has been investigated, carefully delivering them on a circadian schedule has not. Thus, we hypothesize that delivery of these drugs at specific time points within the circadian cycle can enhance bone regeneration.

Materials and Methods:: To measure cell migration, MC3T3-E1 cells were plated in 6-well plates at a concentration of 175,000 cells/well for 4 days in culture medium. A 100nM dexamethasone solution was added to a morning (EST 07:30) and evening group (EST 19:30) on the third day and incubated for 1 hour at 37°C. The wells were washed with PBS and replenished with culture medium. On the fourth day, a scratch assay was performed at EST 13:30, image acquisition was 18 hours after. To measure bone forming potential, MC3T3-E1 cells were plated in 6-well plates at a concentration of 50,000 cells/well for 3 days in culture medium. On days 3, 5, 7, and 9, the application of dexamethasone was as stated above. The wells were washed with PBS and mineralizing medium (α-MEM, 10% FBS, 5% penicillin/streptomycin, 2.304mg/mL B-Glycerol Phosphate and 0.05mg/mL L-Ascorbic Acid) was added. On day 10, a colorimetric and fluorometric Alkaline Phosphatase Assay (ALP) was used to measure osteoblastic activity. To our knowledge, this method has not been used to synchronize MC3T3-E1 cells to a circadian rhythm. Ongoing work consists of adding 20ng/mL of BMP-2 daily along with the dexamethasone protocol as stated above. BMP-2 will be delivered 15 hours after the first application of dexamethasone to match the start of the mineralization phase in the MC3T3-E1 cell line. After 10 days, osteoblastic activity will be measured as stated above.

Results, Conclusions, and Discussions::

Introducing circadian synchronization enhanced cell migration over non-synchronized controls (Figure 1A) as demonstrated by a scratch assay. Dexamethasone treated cells closed the wound almost 20% more than the unsynchronized controls. When measuring bone forming potential, the colorimetric and fluorescence expression (Figure 1B-1C) showed that ALP expression was enhanced following dexamethasone treatment, supporting our hypothesis that cells synchronized to a circadian rhythm can induce faster bone regeneration. Some studies on the effects of dexamethasone in MC3T3-E1 vary, however, our data suggests that dexamethasone at low concentrations and specific times enhances migration and differentiation. BMP-2 is an osteogenic factor that stimulates osteoblast differentiation and bone formation, which leads us to our next steps to investigate the role that BMP-2 plays when added at the optimal circadian time to a circadian synchronized group of MC3T3-E1 cells. Ultimately, we believe that this work will unveil the role of circadian rhythms in bone regeneration and provide us with a new tool to treat these challenging pathologies.

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