Tetracalcium phosphate treatment on experimental fracture model in rats

Abstract

Objectives: This study aims to investigate the efficacy of tetracalcium phosphate (TTCP) on fracture healing in rat femurs.

Materials and methods: Forty-two female Wistar Albino rats were randomized into two groups (Group 1 and Group 2, n=21 for each). The left femur of all animals was fractured by osteotomy after deep anesthesia with ketamine. Additional procedure was not applied to the rats in Group 1. Rats in Group 2, following osteotomy, were applied to the fracture line approximately 2 mL TTCP. The animals were sacrificed at Weeks 1, 2, and 3 after surgery (seven animals were sacrificed from each group each week) and the broken femurs were removed. The femurs were examined first radiographically and second histopathologically.

Results: Radiologically, callus maturity and bone union increased with time in both groups. However, no significant differences were found regarding callus maturity and bone union in weekly comparisons (anteroposterior plain: p=0.53, p=0.37, p=0.42, lateral plain: p=0.26, p=0.42, p=0.87). Histopathologically, the fractures healed normally as the weeks progressed in both groups. The histological scores of Group 2 were higher at Weeks 1, 2, and 3. In the evaluation, no significant difference was found between the groups in terms of histological scores except for the first week (p=0.024, p=104, p=462, respectively).

Conclusion: Although there was no statistically significant difference in the histological evaluation of both groups, except for the first week, the histological scores of Group 2, which underwent TTCP in all weeks, were higher. According to the results of this study, we believe that TTCP may be beneficial, particularly in the early stages of fracture healing.

Figure 1. (a) Fracture formation in the femur and (b) application of TTCP. (c) Confirmation of the postoperative fracture formation by radiological imaging and (d) the radiological appearance of TTCP applied to the fracture site. TTCP: Tetracalcium phosphate.

Figure 2. Radiographic lateral assessment of rat fracture healing femoral radiograms of the rats. (a) Week 1 in Group 2, (b) Week 2 in Group 2, (c) Week 3 in Group 2, (d) Week 1 in Group 1, (e) Week 2 in Group 2, (f) Week 3 in Group 2. Callus maturation and bone union becomes more mature as weeks elapsed.

Figure 3. Radiographic anteroposterior assessment of rat fracture healing femoral radiograms of the rats. (a) Week 1 in Group 2, (b) Week 2 in Group 2, (c) Week 3 in Group 2, (d) Week 1 in Group 1, (e) Week 2 in Group 2, (f) Week 3 in Group 2. Callus maturation and bone union becomes more mature as weeks elapsed.

Figure 4. In comparison of Group 2 and Group 1 within each week of the rats sacrificed at Weeks 1, 2, and 3, Group 2 was found to be higher in histological scoring than Group 1. There was no significant difference between the groups, except for the first week. (a) Group 1, Week 1, sections showing callus consisting entirely of cartilage in Grade V callus formation (H&E, x40). (b) Group 2, Week 1, sections showing callus characterized by equal cartilage and immature bone in Grade VII callus formation (H&E, x40) as well as endochondral bone formation. Red arrow shows calcified trabecular bone and the point of red arrow shows hypercellularity between trabeculae (c) Group 1, Week 2, sections showing immature bone callus with a small amount of cartilage in Grade VIII callus formation (H&E, x40). (d) Group 2, Week 2, sections showing callus characterized by immature bone formation in Grade IX callus formation (H&E, x40). Red arrow shows healing area among minimum gap. White arrow shows new bone matrix and anastomosis of trabeculae. Black arrow shows active osteoblasts synthesizing bone matrix and fusiform progenitor cells (e) Group 1, Week 3, sections showing mature bone formation in a Grade X callus formation (H&E, x10). (f) Group 2, Week 3, sections showing mature bone formation in Grade X callus formation (H&E, x40). It can be seen that early-stage intramembranous ossification occurs. Red arrow shows defect area, hemorrhage, and vanished bone matrix. Blue arrow shows new matrix and lamellar bone formation. T shows old trabeculae.