Advancing Osteoarthritis Care: Unlocking Potential with DMOG and Hyaluronic Acid Treatment

Abstract

Osteoarthritis is a common temporomandibular disease among the adults. Characterized by continues degeneration of chondrocytes which results to altered joint structure and function. The leading approach to cater this issue is to stimulate the regeneration of chondrocytes. In our study we are aiming to stimulate chondrogenesis via a drug called Dimethyl oxalyl glycine associated with Hyaluronic acid gel encapsulating exosomes. Dmog is a Prolyl Hydroxylase inhibitor this mechanism is crucial due to stabilizing hypoxia inducible factor. Prolyl hydroxylase inhibits HIF-1 Alpha. Thus, Dmog stabilizes HIF-1 alpha which results to regulation of chondrocytes genes, increase protein release, and regeneration. Hyaluronic acid (HA) is a naturally occurring polysaccharide that is widely studied for its biological and biomedical applications. Hyaluronic acid will degrade over time which will allow DMOG to gradually stimulate into the targeted site delivered by exosomes. The concentration of hyaluronic acid is crucial for a gradual release of Dmog. Understanding the relationship between concentration and absorbance is vital for accurate quantification and characterization of HA, as well as for evaluating its behavior encapsulating cells, exosomes, and therapeutical drugs. This study aims to investigate the concentration-absorbance relationship of HA through the construction of a standard curve using known concentrations of fluorescent and carbazole assay. It also evaluates chondrocytes gene expressions via QPCR and Immunostaining for protein release and founding that Dmog drug and hypoxic environments are beneficial to induce chondrocyte proliferation and regeneration. The experiment involved four different groups, each consisting of samples with varying concentrations of HA encapsulating mainly 1% and 2%. Also,100nm and 200nm on each concentration respectively. Fluorescent beads mimics the sizes of exosomes used in active practice are encapsulated inside the Hyaluronic acid hydrogels. The carbazole assay was employed to determine the degradation period corresponding to different HA concentrations over a period of time. A series of standard solutions with known concentrations of HA were prepared and subjected to spectrophotometric analysis. The absorbance of each standard solution was measured at a specific wavelength, and a calibration curve was constructed by plotting absorbance against concentration. The resulting standard curve served as a reference to determine the concentration of unknown HA samples based on their absorbance values. Statistical analysis was performed to assess the accuracy and reliability of the method, including the calculation of regression parameters and determination of coefficient of determination (R²) values. In addition, we experimented chondrocytes behaviors under Dmog and Hypoxic environments to evaluate genes expression regulation under physiological hypoxia and chemical hypoxia compared to untreated group. Physiological hypoxia cells were preserved in 5% CO2 hypoxia cabinets, chemical hypoxia had Dmog mixed with media 6% CO2, and the untreated samples had a normal oxygen levels 20% CO2. QPCR provided an insight into genes expression, which could enhance Chondrogenesis. Immunostaining was employed to determine Aggrecan, Col I, and Col II, which are important proteins for Chondrocytes proliferation.In the result, The key findings of the experiments on the relationship between concentration and absorbance in Hyaluronic acid degradation process. The results were visualized using a standard curve. Also, beads release into the supernatant were evaluated using fluorescence assay. Fluorescence beads were used to mimic exosomes in this study used common sizes of exosomes were used 100nm and 200nm. The results clearly demonstrate the observed patterns that suggested 2% concentration of Hyaluronic acid has the optimal potential for a slow degradation rate (7) days, highlighting the importance of understanding the concentration-absorbance relationship in Hyaluronic acid analysis. These findings contribute to our understanding of Hyaluronic acid behavior and its potential applications in various fields. Also, The key findings of QPCR and immunostaining is it provided a positive results and Chondrogenesis responsible genes are elevated while being under Dmog drug and Hypoxic condition compared to Control (untreated) conditions.