Investigation of the efficacy of epidermal growth factor, boric acid and their combination in cartilage injury in rats: An experimental study

Abstract

Objectives: In this study, we aimed to determine the bioefficacy of epidermal growth factor (EGF), boric acid (BA), and their combination on cartilage injury in rats.

Materials and methods: In in vitro setting, the cytotoxic effects of BA, EGF, and their combinations using mouse fibroblast cell (L929), human bone osteosarcoma cell (Saos-2), and human adipose derived mesenchymal stem cells (hAD-MSCs) were determined by applying MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test. In in vivo setting, 72 rats were randomly divided into four groups. A standard chondral defect was created and microfracture was performed in all groups. Group A was determined as the control group. In addition to the standard procedure, Group B received 100 ng/mL of EGF, Group C received a combination of 100 ng/mL of EGF and 10 µg/mL of BA combination, and Group D 20 µg/mL of BA.

Results: The cytotoxic effect of the combinations of EGF dilutions (1, 5, 10, 25, 50, 100, 200 ng/mL) with BA (100, 300, 500 µg/mL) was observed only in the 72-h application period and in Saos-2. The cytotoxic effect of BA was reduced when combined with EGF. There was no significant difference in the histopathological scores among the groups (p=0.13).

Conclusion: Our study showed that EGF and low-dose BA application had a positive effect on cartilage healing in rats. Significant decreases in recovery scores were observed in the other groups. The combination of EGF and BA promoted osteoblast growth. Detection of lytic lesions in the group treated with 20 µg/mL of BA indicates that BA may have a cytotoxic effect.

Figure 1. Stages of surgical experimentation. (a) Image of the experimental animal after general cleaning covering with a sterile drape. (b) Paramedian incision image of the left knee with skin and subcutaneous incision. (c) Image of lateral deviation of the patella and subsequent distal condyles of the femur. (d) Image of the distal condyles of the femur and the defective area. (e) Image after patellar tendon repair. (f) Intra-articular administration of a group-specific active substance.

Figure 2. Microscopic imaging with hematoxylin-eosin (H&E, x10). A-E: Group names, Evaluation weeks 4, 8, and 12.

Figure 3. Microscopic imaging with Masson’s Trichrome (x10). A-E: Group names, Evaluation Weeks 4, 8, and 12.

Figure 4. After (a) boric acid and (b) EGF compounds were applied on L929, Saos-2, and hAD-MSCs at the tested concentration range for 24, 48, and 72 h, the dose-response relationship was determined by MTT cell viability assay (mean ± standard deviation, n=3). EGF: Epidermal growth factor; hAD-MSC: Human amnion-derived mesenchymal stem cell; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 5. The effects of EGF dilutions (1, 5, 10, 25, 50, 100, 200 ng/mL) in combination with BA (100, 300, 500 µg/mL) on cell viability were analyzed by MTT assay after 24, 48 and 72 h of treatment on (a) L929, (c) Saos-2 and (c) hAD-MSCs (mean ± standard deviation, n=3). The DMEM medium containing no compounds was used as a control. EGF: Epidermal growth factor; BA: Boric acid.

Figure 6. Macroscopic Imaging. A-E: Group names, Evaluation Weeks: 4, 8, and 12.

Figure 7. Modified Mankin and O’Driscoll Scoring.