P-071 - Investigating the Mechanism of Dextrose Prolotherapy on Fibroblast and Chondrocyte Cell Viability

Abstract Title: Investigating the Mechanism of Dextrose Prolotherapy on Fibroblast and Chondrocyte Cell Viability


Background: The limited vascular nature of ligaments and cartilage can lead to suboptimal healing from injury that may result in joint laxity or osteoarthritis (OA). OA is the most prevalent articular disease worldwide¹ and current recommended treatment focuses on symptom management until severity increases requiring joint replacement. In 1958, Dr. George Hackett demonstrated ligaments could be strengthened by injection with a proliferative agent². Many proliferative agents have been used since then; however, 12.5% and 25% dextrose prolotherapy are particularly popular due to its relatively low cost and minimal side effect profile³. Prolotherapy is thought to induce an initial inflammatory response surrounding the injection site, stimulating cellular proliferation and tissue repair, ultimately resulting in improved strength and stability. Increased cartilage thickness, decreased pain, and improved stability following prolotherapy treatment has been observed in patients with knee OA and other joint disorders^4-6. However, cell-based studies investigating the mechanism of prolotherapy are limited and clinical trials have differed greatly in the injection procedure, resulting in varied outcomes^3,7-11.

Purpose/Objectives: Dextrose prolotherapy injections represent a promising non-surgical treatment option for patients with OA. However, more research is needed for dextrose prolotherapy to become a standard, recommended treatment¹¹. The mechanism of dextrose prolotherapy is also not well understood, and additional studies are needed to determine the optimal dextrose concentration. The goal of this study is to evaluate how fibroblasts and chondrocytes respond following dextrose exposure as a method to explore the potential cellular mechanism of dextrose prolotherapy.

We hypothesize that hypertonic solutions of 10-25% dextrose will incite an initially damaging, inflammatory response followed by the production of growth factors and enhanced cellular proliferation.


Methods: In the first experiment, MRC- 5 Fibroblasts were exposed to varying concentrations of dextrose (5, 10, 15, 20, 25%) or a media control without additional dextrose for 15, 30, 60, or 120 minutes. After reaching the appropriate timepoint, an XTT assay was used to determine cell viability. Samples were run in duplicate from five independent experiments.

In the second experiment, MRC- 5 Fibroblasts were exposed to varying concentrations of dextrose (5, 10, 15, 20, 25%) or a media control without additional dextrose for 15, 30, 60, or 120 minutes. After reaching the appropriate time point, the supernatant fluid containing dextrose or the control was removed and replaced with fresh media; the fibroblasts were then incubated at 37°C, 5% CO2. 48 hours later an XTT assay was used to determine cell viability. Samples were run in triplicate from five independent experiments.

In the third experiment, the first experiment type was repeated using CHON-001 Chondrocytes. Samples were run in duplicate from four to five independent experiments.

All results were placed into a mixed effect model and analyzed using ANOVA to compare control vs treatments to determine significance.

Results: When evaluating the viability of fibroblasts immediately after being treated with dextrose, we found that higher concentrations of dextrose solutions or longer treatment times reduced cell viability. ANOVA analysis of fibroblasts treated directly with 10% and 15% dextrose for 120 minutes, 20% dextrose for 60 minutes or longer, and 25% dextrose for 15 minutes or longer significantly reduced cell viability (p < 0.05) compared to the media control. However, the fibroblasts exposed to 20% dextrose for 60 minutes recovered 48 hours post treatment (p< 0.05). Alternatively, data from chondrocytes indicate that they may respond differently to dextrose exposure. Immediately after chondrocytes were exposed to 20% dextrose for 60 minutes, there was an increase in cell viability (p< 0.05) while exposure to 20% or 25% dextrose at 120 minutes decreased cell viability (p< 0.05).

Overall, direct exposure to hypertonic solutions of dextrose ( >5%) for 120 minutes will decrease cell viability in fibroblasts and those fibroblasts are not able to recover 48 hours post treatment. Fibroblasts exposed to dextrose concentrations < 25% for 60 minutes or less recover by 48 hours post treatment. Direct exposure to higher concentrations (20 & 25%) of dextrose for 120 minutes will decrease cell viability in chondrocytes. The results show fibroblasts appear to be more sensitive to dextrose exposure compared to chondrocytes.

Conclusions/Implications for future research and/or clinical care: The results of our study support pieces of the proposed hypothesis for dextrose prolotherapy. It showed that fibroblasts were initially damaged by exposure to dextrose but then were able to recover post-treatment. However, our in vitro cellular model does not account for the cellular heterogeneity of the joint space. In vivo injections of dextrose into the joint space may result in a variety of cell types being exposed to varying and changing concentrations of dextrose. Further research is needed to identify the optimal therapeutic dextrose concentrations and precise mechanism of the therapy. Additionally, analysis of factors secreted in response to the prolotherapy treatment is needed to continue to explore and elucidate the mechanism. Further research and information provide the potential for continuing improvement in clinical outcomes and may lead to evidence-based recommendations for the use of dextrose prolotherapy as an effective, minimally invasive, affordable, nonsurgical option for patients with OA.

References: 1. Maqbool M, Fekadu G, Jiang X, et al. An up to date on clinical prospects and management of osteoarthritis. Ann Med Surg (Lond). 2021;72:103077. Published 2021 Nov 19. doi:10.1016/j.amsu.2021.103077

2. Hackett, G.S. Ligament and Tendon Relaxation. 3rd ed. Charles C Thomas; 1958.

3. Reeves KD, Sit RWS, Rabago DP. Dextrose Prolotherapy: A Narrative Review of Basic Science, Clinical Research, and Best Treatment Recommendations. Phys Med Rehabil Clin N Am. 2016;27(4):783-823. doi:10.1016/j.pmr.2016.06.001

4. Topol GA, Podesta LA, Reeves KD, et al. Chondrogenic Effect of Intra-articular Hypertonic-Dextrose (Prolotherapy) in Severe Knee Osteoarthritis. PM&R. 2016;8(11):1072-1082. doi:10.1016/j.pmrj.2016.03.008

5. Rabago D, Kansariwala I, Marshall D, Nourani B, Stiffler-Joachim M, Heiderscheit B. Dextrose Prolotherapy for Symptomatic Knee Osteoarthritis: Feasibility, Acceptability, and Patient-Oriented Outcomes in a Pilot-Level Quality Improvement Project. J Altern Complement Med. 2019;25(4):406-412. doi:10.1089/acm.2018.0361

6. Sit RWS, Reeves KD, Zhong CC, et al. Efficacy of hypertonic dextrose injection (prolotherapy) in temporomandibular joint dysfunction: a systematic review and meta-analysis. Sci Rep. 2021;11:14638. doi:10.1038/s41598-021-94119-2

7. Woo MS, Park J, Ok SH, et al. The proper concentrations of dextrose and lidocaine in regenerative injection therapy: in vitro study. Korean J Pain. 2021;34(1):19-26. doi:10.3344/kjp.2021.34.1.19

8. Ekwueme EC, Mohiuddin M, Yarborough JA, et al. Prolotherapy Induces an Inflammatory Response in Human Tenocytes In Vitro. Clin Orthop Relat Res. 2017;475(8):2117-2127. doi:10.1007/s11999-017-5370-1

9. Güran Ş, Çoban ZD, Karasimav Ö, et al. Dextrose solution used for prolotherapy decreases cell viability and increases gene expressions of angiogenic and apopitotic factors. GULHANE Med J. 2018;60(2):42. doi:10.26657/gulhane.00016

10. Freeman JW, Empson YM, Ekwueme EC, Paynter DM, Brolinson PG. Effect of prolotherapy on cellular proliferation and collagen deposition in MC3T3-E1 and patellar tendon fibroblast populations. Transl Res. 2011;158(3):132-139. doi:10.1016/j.trsl.2011.02.008

11. Kolasinski SL, Neogi T, Hochberg MC, et al. 2019 American College of Rheumatology/Arthritis Foundation Guideline for the Management of Osteoarthritis of the Hand, Hip, and Knee. Arthritis Rheumatol. 2020;72(2):220-233. doi:10.1002/art.41142