Hi, my name is Connor Kroll and I'm a third year medical student at Campbell. I'm a part of a team researching dextrose prolotherapy, which is in the field of regenerative medicines. Ligaments and cartilage are often a focus of this field because of their avascular nature, which leaves them with limited healing capabilities, ultimately leading to laxity and osteoarthritis. Dextrose is a commonly used agent because of its inexpensive cost and minimal side effects. Cellular solutions injected into a joint or ligament are proposed to cause an initial irritating and inflammatory response, leading to cellular recruitment and proliferation ultimately with the hopes of tissue repair and increased joint stability. Our own cellular studies and others have shown increased cellular response, proliferation and cytokine recruitment at the clinical prolotherapy dextrose doses of 12.5 to 20%. However few cellular studies have investigated the second most commonly included substance in dextrose prolotherapy, analgesic agents for pain control. Lidocaine is most commonly used and has been shown in a variety of cell studies to be both cytostatic and cell cytotoxic. Our own preliminary studies found that lidocaine may actually have a synergistic effect with dextrose on cellular proliferation stage of prolotherapy. For these reasons, we continue to investigate how lidocaine may affect dextrose prolotherapy. We used a human fibroblast line to test our hypothesis that cells treated with solutions containing both dextrose and lidocaine will secrete pro-inflammatory and proliferative cytokines that can then be used on nascent fibroblasts to enhance proliferation compared to fibroblasts treated with dextrose alone. So our methods. The human fibroblast line shown in figure 2 is a Merck 5 lung fibroblast. They were grown in 96-volt plates to prepare for our experiments. Our first protocol, while is included in this talk, is not the focus of our hypothesis. However, we felt that it would give a better understanding into our preliminary results. In our first protocol, we directly exposed these fibroblast cells to dextrose and lidocaine. Cells were treated using either a media control, dextrose only control, or a dextrose plus a varying concentration of lidocaine from 0.1 to 1% lidocaine for exposure times from 15 to 2 hours. The cells following their exposure to the fibroblast were then analyzed using the cytoquant XTT assay, which uses metabolic activity as an indicator of cell viability. The XTT assay, shown on the 96-volt plate in figure 3, undergoes a color change which represents cell metabolism, which then can be directly correlated to cell viability. So increased color change represents an increase in cellular viability. In our second protocol, we tested the indirect influence of dextrose-lidocaine solutions. Cells were treated exactly the same as they were in the first protocol, except for the duration of exposure time. It was from 15 to 60 minutes. After full exposure time points were met, the treatment solutions were removed and replaced with fresh media, and then maintained for 8 hours to collect any secreted factors. After 8 hours, the media was then taken off, and the supernatant fluid was removed and placed onto nascent fibroblasts that had never been exposed to treatment solutions before. These nascent fibroblasts were then maintained in the supernatant fluid for 48 hours, after which an XTT assay was performed. This allowed us to see how cells in a joint that are not directly exposed to dextrose may respond. So, the results. So if you can see here, in all of these graphs, you can see that the x-axis is the absorbance, which is the measure of the XTT assay, and the y-axis is our time. When increased absorbance values equals increased cell viability, the various color bars represent the different concentrations of treatment, and the stars denote significance. So in figure 7, as previously mentioned, figure 7 is our prior preliminary data, but it should help with an understanding of lidocaine's effect on dextrose prolotherapy. These results show that higher concentrations of lidocaine for longer exposure times has a more deleterious effect on cell viability compared to dextrose alone, however moderate doses such as 0.5% lidocaine for shorter exposure periods increase cell viability compared to dextrose alone. The results from our second protocol, investigating the indirect effects of dextrose, are shown in figure 8 and 9. Figure 8 represents a moderate concentration of dextrose at 12.5, while figure 9 represents a higher dose of dextrose at 20%. We see the original dextrose treatment time on the x-axis, and the absorbance measure from the XCT assay of nascent fibroblast on the y-axis. The results in both figure 8 and 9 support our hypothesis that the addition of lidocaine would result in more secreted factor generation and higher proliferation in nascent fibroblast untreated cells over dextrose alone. However, compared to our first protocol, it seems that the cells treated with higher concentrations of lidocaine for longer exposure times produce more secreted factors which ultimately result in better viability of nascent untreated cells. So what does this tell us? The results from our experiment support our hypothesis that the addition of lidocaine to dextrose prolotherapy may result in a greater initial inflammatory and damaging response, but then this stimulates the production of growth factors in cellular proliferation when compared to dextrose alone. It is important to note that our cellular study has limitations due to its difficulty in replicating the environment of the joint space, however much dextrose or lidocaine may be diluted once it enters the joint, and how long these substances may persist in the joint once they are injected. Ultimately, more research is needed to inform the optimal concentrations for use clinically and the precise mechanism of dextrose prolotherapy. Continued research and information provide the potential for improved clinical outcomes and may lead to evidence-based recommendations for the use of dextrose prolotherapy as an effective minimally invasive non-surgical option for patients with osteoarthritis. When we think about prolotherapy overall, we see its potential for a treatment that is based around promoting the body's self-healing to improve both structure and function. I apologize, and thank you so much for listening to my talk, I hope you have a good day!

dextrose prolotherapy

regenerative medicine

osteoarthritis

lidocaine

tissue repair