Characterization of a rat osteotomy model with impaired healing

Abstract

Background: Delayed union or nonunion are frequent and feared complications in fracture treatment. Animal models of impaired bone healing are rare. Moreover, specific descriptions are limited although understanding of the biological course of pathogenesis of fracture nonunion is essential for therapeutic approaches.

Methods: A rat tibial osteotomy model with subsequent intramedullary stabilization was performed. The healing progress of the osteotomy model was compared to a previously described closed fracture model. Histological analyses, biomechanical testing and radiological screening were undertaken during the observation period of 84 days (d) to verify the status of the healing process. In this context, particular attention was paid to a comparison of bone slices by histological and immunohistological (IHC) methods at early points in time, i.e. at 5 and 10 d post bone defect.

Results: In contrast to the closed fracture technique osteotomy led to delayed union or nonunion until 84 d post intervention. The dimensions of whole reactive callus and the amounts of vessels in defined regions of the callus differed significantly between osteotomized and fractured animals at 10 d post surgery. A lower fraction of newly formed bone and cartilaginous tissue was obvious during this period in osteotomized animals and more inflammatory cells were observed in the callus. Newly formed bone tissue accumulated slowly on the anterior tibial side with both techniques. New formation of reparative cartilage was obviously inhibited on the anterior side, the surgical approach side, in osteotomized animals only.

Conclusion: Tibial osteotomy with intramedullary stabilisation in rats leads to pronounced delayed union and nonunion until 84 d post intervention. The early onset of this delay can already be detected histologically within 10 d post surgery. Moreover, the osteotomy technique is associated with cellular and vascular signs of persistent inflammation within the first 10 d after bone defect and may be a contributory factor to impaired healing. The model would be excellent to test agents to promote fracture healing.

Figure 1

(a-h). Radiographs of a rat tibia after osteotomy (a-d) or fracture (e-h) over the complete observation period: (a and e) day of surgery, (b and f) 28 d post surgery, (c and g) 42 d post surgery and (d and h) 84 d post surgery. Although there was the best possible fixation and there were no signs of osteolysis, radiographs of osteotomized animal revealed extremely impaired healing and clearly indicated nonunion.

Figure 2

(a and b). Safranin O/von Kossa staining to detect mineralized bone (black staining) of comparable areas in representative tibiae 84 d after osteotomy (a) and fracture (b) and intramedullary stabilization demonstrating nonunion and regular healing respectively. Cavities correspond to the 1 mm wire diameter. Asterisks indicate the proximal and anterior tibial side.

Figure 3

(a and b). HE stained slices of comparable osteotomized (a) and fractured tibia areas (b) 10 d after surgery. The anterior tibial side is located at the bottom of pictures; diamonds indicate the anterior tibial side. Cavities correspond to the 1 mm wire diameter.

Figure 4

(a and b). Micrographs of representative cellular types in callus adjacent to the bone defect after 10 d. Apart from fibrozytes and fibroblasts (arrows), osteotomized animals accumulate large numbers of lymphocytes, granulocytes and multinucleated giant cells (arrowheads). In contrast, callus in fractured animals hardly contained any inflammatory cells (b). HE staining, magnification 200×.

Figure 5

Immunohistological staining of α-smooth muscle actin for selective staining of blood vessels (red labeled) in the callus of an osteotomized animal. Magnification 200×.

Figure 6

(a and b). Histological preparation of comparable osteotomized (a) and fractured (b) tibial areas 10 days after surgery. Movat Pentachrome staining for detection of newly formed trabecular bone (arrows). Cavities correspond to the 1 mm wire diameter. Diamonds indicate the proximal and anterior tibial side.

Figure 7

(a and b). Histological preparation of an osteotomized (a) and fractured (b) tibia 10 days after surgery. Alcian blue staining for cartilage to detect hyperblastic zones of chondrocytes. Diamonds indicate the proximal and anterior tibial side. Note the homogeneous pattern of cartilage around the fractured fibula compared to the osteotomized tibia in the same animal (a). Cavities correspond to the 1 mm wire diameter.