The effect of theranekron on femur fracture healing in an experimental rat model

Abstract

Objectives: The aim of this study was to investigate the radiological, biomechanical, histopathological and immunohistochemical effects of theranekron on fracture healing in an experimental rat model.

Materials and methods: Forty-eight male albino Wistar rats were used. Four groups were formed, with 12 rats in each of theranekron groups 1 and 2, and control groups 1 and 2. After a fracture was created in the right femur of the rats included in the study, fixation was performed with an intramedullary Kirschner wire. Theranekron was administered subcutaneously to theranekron groups 1 and 2 at a dose of 0.3 mg/kg on days 0, 5 and 10. After radiographic analysis of the femurs of theranekron group 1 and control group 1 rats at four weeks of the study was performed, both groups were divided into two equal subgroups (six femurs in each group). Histopathological and immunohistochemical examinations were performed in one subgroup and biomechanical examination in the other subgroup. At the end of six weeks, the rats in theranekron group 2 and control group 2 were evaluated after applying the same procedure as in the fourth week.

Results: When the mean radiological scores of the theranekron and control groups were compared, a statistically significant difference was found in favor of the theranekron group at four and six weeks (p=0.028 and p=0.006, respectively). At four weeks, statistically significant higher biomechanical forces were obtained in the theranekron group compared to the control group (p=0.030). In the histopathological evaluation, the inflammation value of the control group at four weeks was statistically significantly higher than the theranekron group (p=0.027). The angiogenesis, osteoblast proliferation, and bone formation values of the theranekron group were significantly higher than the control group (p=0.014, p=0.014, and p=0.005, respectively). At six weeks, the bone formation values of the theranekron group were statistically significantly higher than the control group (p=0.021). The difference between the theranekron group and the control group scores of the immunohistochemical evaluation were statistically significantly different at four and six weeks (p=0.006 and p=0.011, respectively).

Conclusion: Theranekron may play a role in accelerating fracture healing by reducing acute inflammation process in the early period of fracture union, increasing fracture strength, angiogenesis, osteoblast proliferation, and bone formation.

Figure 1. (a) Standard transverse osteotomy line. (b) Intramedullary insertion of the K-wire into the femur.

Figure 2. Radiographic confirmation of fracture and fixation.

Figure 3. Details of the three-point bending test.

Figure 4. Evaluation of fracture healing at 4th and 6th weeks, Theranekron group 1, 2 (a, c), Control group 1, 2 (b, d).

Figure 5. (a) Control, 4th week. In the fracture area, prominent purulent (Pr) exudate is observed in the areas close to the center of the femur, and angiogenesis (Ag) and fibrosis (F) are observed just at the periphery of this inflammatory exudate. H.E. Bar; 200 µm. (b) Control, 4th week. Angiogenesis (A), fibrosis (F), proliferation of diffuse chondroblastic (CHB) cells, cartilage (chondrocyte) and osteoid tissue formation are observed in the areas close to the center of the femur in the fracture area. H.E. Bar; 500 µm. (c) Theranekron, 4th week. Angiogenesis (A), mild infiltration of inflammatory cells and fibrosis (F) are observed in the areas close to the center of the femur in the fracture area, while proliferation of diffuse chondroblastic (CHB) cells (cartilage tissue formation) is observed in the periphery. H.E. Bar; 500 µm. (d) Theranekron 4th week. Angiogenesis (Ag), fibrosis (F) and prominent proliferation of osteoblastic cells (OSB) are observed in the periphery of newly formed osteoid (OSD) tissue at the fracture site. H.E. Bar; 100 µm. (e) Control, 6th week. While fibrosis (F), angiogenesis (Ag) and mildly purulent (Pr) exudate are observed in the fracture area near the center of the femur; significant proliferation of osteoblastic cells, formation of bone trabeculae (TB) with a small amount of bone marrow, and proliferation of chondroblastic (CHB) cells (cartilage tissue formation) are observed in more peripheral areas. Note that the regeneration is not yet completed. H.E. Bar; 500 µm. (f) Theranekron, 6th week. Bone trabeculae, including bone marrow, are observed at the fracture site. Note that the regeneration is completed. H.E. Bar; 500 µm

Figure 6. Osteopontin expression. While a weak reaction (a) was observed in the regeneration region of control group rats in the 4th week, a more pronounced and more widespread reaction was observed in the (b) Theranekron group. (c) In control rats in the 6th week, weak staining was observed in osteoblasts in the regeneration zone. (d) In the Theranekron group, a very distinct and more widespread immunoreaction was observed. Immunperoxidase staining, Bar; 100 µm.