Experimentally created mid diaphyseary fractures in 24 rabbits were treated by intramedullary pinning with K-wires, PMMA implants and intramedullary pinning after stuffing with HA paste and the histopathological changes were recorded. Healing process was not progressive, in control group, as indicated by presence of only a few osteocytes in the matrices, discontinuity in the epithelial layer and extensive vacuolation with occasional presence of giant cells. In PMMA group, healing pattern by endochondral method of ossification was evident by day 30 and by day 60, normal structure of bone with remarkably high osteoid formation and osteocytes proliferation were observed. In HA group, vacuolation and discrete presence of osteocytes separated by interlacing trabeculae when compared to the other two groups were more pronounced. By day 60, endosteal vascularization could be depicted under high power. To conclude, of all the three groups healing was better in PMMA group when compared to the other two groups.
Fracture is defined as a discontinuity in hard tissues such as bone and cartilage. The fracture in animals invariably causes pain and suffering to them, apart from loss of function of’ the affected limb. The method to address fractures in different species varies significantly. In the recent years, there has been a trend to use biodegradable and bio-absorbable implants for fracture healing in animals. As poly methyl methacrylate (PMMA) can be molded to different shapes and sizes, it was used for the repair of fractures (Cho and Gosain, 2002). Hydroxyapatite is a biocompatible implant material having osteoconductive capacity (Craig et al., 1989) and is known for its passive support for neovascularization (Borges et al., 1998). In the present paper, the efficacy of PMMA and HA was shown in terms of histopathological features.
Materials and Methods
The experiment was conducted in twenty-four rabbits divided in to three groups of eight each. In all the animals, diaphyseary fractures were created using a circular wire saw and were immobilized with K-wires (n=8; Group- I), PMMA implants (n=8; Group, II) and HA quoted implants (n=8; Group, III). The stainless-steel K-wires of 1-2 diameter were employed to immobilize the fracture fragments through standard protocol. In the second group, the PMMA implants resembling K-wires were prepared by mixing the powder with liquid copolymer and were used to immobilize the fractur fragments (Fig. 1). It was smeared in the groove of a mold under low temperature and the implant was used for immobilizing the fracture fragments. In the third group, HA paste was stuffed in to the medullary cavity of the fragments and K-wires were used for fixation (Fig. 2).
|Fig. 1: Note PMMA implant immobilizing the fracture fragments||Fig. 2: Note HA paste stuffed in to the medullary cavity of bone fragments.|
Two rabbits from each group were euthanized using thiopentone sodium by intravenous route till effect and the fractured femur was collected for gross and histopathological evaluation. The bone samples were made free from the soft tissue. After total decalcification, bone samples were subjected for routine paraffin embedding technique and the sections were stained by the Hematoxylin-Eosin staining technique as per the method of Singh and Sulochana (1997).
Results and Discussion
Healing process was not progressive after 15 days, in control group, as indicated by complete loss of normal histopathological structure with only a few osteocytes in the matrices discontinuities of the epithelial layer and extensive vacuolation with occasional presence of giant cells were recorded (Fig. 3). In one animal, that developed osteomyelitis the corresponding sections exhibited edema of the dense connective tissue with vacuolation (Fig. 4).
|Fig. 3: Note unorganized osteoid and a very few osteocytes in control group, day 30 (H&E, 10X).||Fig. 4: Note osteomyelitis with edema of dense connective tissue (H&E, 10X).|
In PMMA group, on day 15, sections revealed dense connective tissue with comparatively less number of osteocytes and intact layer of fibrous part of periosteal layer (Fig. 5), healing pattern by endochondral method of ossification was evident by day 30 (Fig. 6).
|Fig. 5: Note intact layer of fibrous portion of periosteal layer in PMMA group, day 30 (H&E, 10X).||Fig. 6: Note endochondral method of ossification in PMMA group, day 30 (H&E, 10X)|
There was an exaggerated growth of epithelium with matrices in the bone cortex containing chondrocytes. At high power, active osteocyte proliferation was also evident sub-periosteally. By day 60, sections revealed normal structure of bone with remarkably high osteoid formation and osteocytes proliferation (Fig. 7). In HA group, vacuolation and discrete presence of osteocytes separated by interlacing trabeculae when compared to the other two groups were more pronounced (Fig. 8). By day 60, osteogenic activity through endochondral ossification was found in one section with complete epithelialization with endosteal vascularization could be depicted under high power (Fig. 9). At 30 days, perfect union of fracture fragments was not achieved and the callus was rubber like without mineral deposition in control group.
|Fig. 7: Note near normal bone structure in PMMA group, day 60 (H&E, 10X).||Fig. 8: Note pronounced vacuolation and predominant osteocyte formation in HA group, day 30 (H&E, 10X).|
In PMMA group, healing was superior with the presence of a fibrous callus around the fracture line. In HA group, though there was presence of a primary callus, periosteal layer was not seen. These findings were contradicting with Na et al. (2012) who observed that the fracture line could not be identified by 4th week after experimental creation of metaphyseal fracture.
|Fig. 9: Photograph showing endosteal vascularization in HA group, day 60.||Fig.10: Photograph showing weight bearing on the hind limb after recovery.|
At 60 days, cicatrization was noticed at the fracture site in control group and unresorbed callus was found. In PMMA group, the healed bone sample almost resembled the normal bone. In hydroxyapatite group, excessive cicatrization and excessive connective tissue growth were found. These results were in accordance with those of Saraf et al. (2007), who observed incomplete union and soft callus 4 weeks after surgery. In PMMA group, there was epithelialization and fibrous tissue formation in the periosteal layer which was in accordance with the findings of Miller et al. (1976) and Ito (1991). In PMMA group, histopathological sections clearly revealed healing pattern by endochondral method of ossification with presence of large number of osteocytes. Figueiredo et al. (2004) reported the differentiation of connective tissue cells in to chondroblasts and osteoblasts in implants of demineralized bone matrix.
Contrary to the findings of Flately et al. (1983), complete healing with osteoid formation was seen in half of the control animals. In PMMA group, concentric layers of bone lamellae with giant cells indicative of inflammatory reaction were noted as recorded by Togawa et al. (2003) and Verlaan et al. (2004). No giant cells were found in hydroxyapatite group which is in accordance with the findings of Kato et al. (1979), Holmes (1979), Braz et al. (2003) and Carlo et al. (2009). Absence of fibrous tissue between implant and bone in hydroxyapatite group was in accordance with findings of Uchida et al. (1990) but contradicting with the findings of Alibadi et al. (2012). Total bone formation was more in hydroxyapatite group when compared to control group which is in accordance with the findings of Lima et al. (2007).
To conclude, of all the three groups healing was better in PMMA group when compared to the other two groups. K-wires, PMMA and HA implants were employed to treat experimentally created mid-diaphyseary fractures in rabbits and were sacrificed at different time intervals to judge their suitability histopathologically.