Korean J Pain.  2021 Jan;34(1):19-26. 10.3344/kjp.2021.34.1.19.

The proper concentrations of dextrose and lidocaine in regenerative injection therapy: in vitro study

• Woo MS
• Park J
• Ok SH
• Park M
• Sohn JT
• Cho MS
• Shin IW
• Kim YA

• KMID: 2510308
• DOI: http://doi.org/10.3344/kjp.2021.34.1.19

Abstract

Background
Prolotherapy is a proliferation therapy as an alternative medicine. A combination of dextrose solution and lidocaine is usually used in prolotherapy. The concentrations of dextrose and lidocaine used in the clinical field are very high (dextrose 10%-25%, lidocaine 0.075%-1%). Several studies show about 1% dextrose and more than 0.2% lidocaine induced cell death in various cell types. We investigated the effects of low concentrations of dextrose and lidocaine in fibroblasts and suggest the optimal range of concentrations of dextrose and lidocaine in prolotherapy.
Methods
Various concentrations of dextrose and lidocaine were treated in NIH-3T3. Viability was examined with trypan blue exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Migration assay was performed for measuring the motile activity. Extracellular signal-regulated kinase (Erk) activation and protein expression of collagen I and α-smooth muscle actin (α-SMA) were determined with western blot analysis.
Results
The cell viability was decreased in concentrations of more than 5% dextrose and 0.1% lidocaine. However, in the concentrations 1% dextrose (D1) and 0.01% lidocaine (L0.01), fibroblasts proliferated mildly. The ability of migration in fibroblast was increased in the D1, L0.01, and D1 + L0.01 groups sequentially. D1 and L0.01 increased Erk activation and the expression of collagen I and α-SMA and D1 + L0.01 further increased. The inhibition of Erk activation suppressed fibroblast proliferation and the synthesis of collagen I.
Conclusions
D1, L0.01, and the combination of D1 and L0.01 induced fibroblast proliferation and increased collagen I synthesis via Erk activation.

Keyword

Actins; Cell Migration Assay; Cell Proliferation; Collagen Type 1; Extracellular Signal-Regulated MAP Kinases; Fibroblast; Glucose; Lidocaine; Muscle; Smooth; Prolotherapy

Figure

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  Fig. 1 Trypan blue exclusion assay after treating the various concentrations of lidocaine and dextrose for 24 hours in NIH-3T3. Cell viability after treating various concentrations of lidocaine (A), and dextrose (C), and inverted microscopic images, respectively (B: lidocaine, D: dextrose). Medians and standard deviation, n = 8 for each group. Ctrl: control. *P < 0.05 and ***P < 0.001.

• 

  Fig. 2 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay after treating 0.01% and 0.05% of lidocaine (A), dextrose (B), and 0.01% of lidocaine (L0.01) and 1% of dextrose (D1) and combination treatment of L0.01 + D1 (C) for 24 hours. Medians and standard deviation, n = 8 for each group. Ctrl: control. ***P < 0.001.

• 

  Fig. 3 Lidocaine and dextrose increase the motile activity of fibroblast. Migration assay after treating L0.01, D1, and L0.01 + D1 for up to 24 hours. (A) Inverted microscopic image for quantifying the cell free area (scale bar = 100 μm), and (B) quantified data for cell free area using the ImageJ. Medians and standard deviation, n = 3 for each group. Ctrl: control. **P < 0.01 and ***P < 0.001.

• 

  Fig. 4 Lidocaine and dextrose activate the extracellular signal-regulated kinase (Erk) signaling pathway and increase the collagen I and α-smooth muscle actin (α-SMA) synthesis. Representative data of western blotting for Erk1/2 and phospho-Erk1/2 (p-Erk1/2) after treating L0.01, D1, and L0.01 + D1 during 3 hours (A), relative expression level of Erk1/2 for β-actin (B), relative expression level of p-Erk1/2 for β-actin (C), representative data of western blotting for collagen I and α-SMA during 24 hours (D), relative expression level of collagen I for β-actin (E), and relative expression level of α-SMA for β-actin (F). Medians and standard deviation, n = 3 for each group. Ctrl: control. *P < 0.05 and **P < 0.01.

• 

  Fig. 5 Inhibition of extracellular signal-regulated kinase (Erk) phosphorylation suppresses the fibroblast proliferation and collagen I synthesis. (A) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay after treating L0.01, D1, and L0.01 + D1 with Erk inhibitor (PD98059; 5 μM and 10 μM) for 24 hours, n = 7. (B) Representative data of western blotting for Erk1/2, phospho-Erk1/2 (p-Erk1/2), and collagen I expression after treating L0.01, D1, and L0.01 + D1 with PD98059 10 μM during 3 hours, n = 3. Medians and standard deviation. Ctrl: control. ***P < 0.001.

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