BackgroundThe use of a closed fracture model has become the preferred model to study the fracture healing process, given that the periosteum and the soft tissue surrounding the fracture site play an important role in the fracture healing process. Some techniques like osteotomy, drilling the long bones and the use of the guillotine-like apparatus to induce fracture are characterized by some undesirable effects and complications. The aim of this study is to optimize and evaluate an in vivo fracture model using three-point bending pliers that can be used to study secondary bone fracture healing in rats.MethodsModified three-point bending pliers were used as a device to create the closed rat tibial bone fracture that was prefixed with an intramedullary pin (23 G × 11/2″) in rats. The exact location of the induced closed fracture was along the long bone. The presence of bone comminution, and the fracture bone alignment were immediately examined after the induction of the fracture until the 6th week.ResultsAll fractures induced were transverse, located in the middle to proximal one third of the tibia, and they all healed without complications. Bone union as shown radiographically occurred within 2–3 weeks postoperative. The average angle of the fracture line with the axis of the tibia was 89.41 ± 2.11°. The lateral and anterio-posterior pin angulation views were 167.33 ± 3.67° and 161.60 ± 4.87° respectively. The average length of proximal end of the fractured bone in comparison with the whole length of intact bone was 41.02 ± 3.27%. There was a significant difference in percentage of the gross callus area and gross callus index, while there was no significant difference in X-ray callus index. There was no significant difference of the gross callus area between slight comminution (n = 4) and non comminution (n = 21).ConclusionThe optimized rat tibial fracture model resulted in mainly transverse tibial mid-shaft fractures with minimal bone comminution and absence of surrounding soft tissue damage. The size area of consequent soft callus formation and the extent to which the closed fracture model was reproducible are very good outcomes making it feasible for in vivo laboratory research use.

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Background Chondrocytes in the growth plate ( GP ) undergo increases in volume during different cascades of cell differentiation during longitudinal bone growth. The volume increase is reported to be the most significant variable in understanding the mechanism of long bone growth. Methods Forty‐five postnatal Sprague‐Dawley rat pups, 7‐15 days old were divided into nine age groups (P7‐P15). Five pups were allocated to each group. The rats were sacrificed and tibia and metatarsal bones were harvested. Bone lengths were measured after 0, 24, 48, and 72 hours of ex vivo incubation. Histology of bones was carried out, and GP lengths and chondrocyte densities were determined. Results There were significant differences in bone length among the age groups after 0 and 72 hours of incubation. Histological sectioning was possible in metatarsal bone from all age groups, and in tibia from 7‐ to 13‐day‐old rats. No significant differences in tibia and metatarsal GP lengths were seen among different age groups at 0 and 72 hours of incubation. Significant differences in chondrocyte densities along the epiphyseal GP of the bones between 0 and 72 hours of incubation were observed in most of the age groups. Conclusion Ex vivo growth of tibia and metatarsal bones of rats aged 7‐15 days old is possible, with percentage growth rates of 23.87 ± 0.80% and 40.38 ± 0.95% measured in tibia and metatarsal bone, respectively. Histological sectioning of bones was carried out without the need for decalcification in P7‐P13 tibia and P7‐P15 metatarsal bone. Increases in chondrocyte density along the GP influence overall bone elongation.

In secondary fracture healing, callus proliferate, undergo hypertrophy and the extracellular matrix becomes calcified. This step to some extent, recapitulates the embryological bone development with a combination of cellular proliferation and differentiation, increasing cellular volume and matrix deposition. The causes of the chondrocytes volume increase in secondary bone healing are poorly known, but cell membrane transporters perhaps could be implicated. We hypothesize that NHE-1 and AE-2 are among plasma membrane transporters that have a role in cellular differentiation and regulation of endochondral ossification for secondary bone fracture healing. Study of closed tibia fracture healing in 2 groups of 25 of 8-weeks-old Sprague-Dawley rats were undertaken and histological evaluation were made at 5 different time points at 1, 2, 3, 4, and 6 weeks after induction of the fracture. Histological evaluation of proliferative and hypertrophic chondrocyte zone area showed a significant difference in week 1 compared to other weeks. Immunohistochemistry study revealed a significant high level of labeling intensity of NHE-1 at the first four weeks. While labeling intensity of AE-2 showed moderate reaction at 1 and 2 weeks, that increased and reached the highest level at 3 and 4 weeks. These results suggested that NHE-1 and AE-2 had role in the endochondral ossification of secondary bone healing.

A fracture model in rats for the study of secondary bone healing was described. Standard open midshaft transverse metatarsal fracture was produced with bone cutting forceps in 28 rats. The commonly open and close fracture models utilized for bone and mineral researches are associated with varying degree of complications ranging from a high degree of fracture comminution to severe associated soft tissue injury which interferes with the healing process. We hypothesized that fracture model in rat third metatarsal bone could be associated with low -post-surgical complications and could be a reproducible model. To test this, open mid-shaft transverse fractures were created on the metatarsals of 28 rats. The objectives of the study were to evaluate the fracture complications, to determine the nature of fracture produced, evaluate the fracture consolidation during healing periods, and to assess the histological and radiographic healing of the fracture. The fracture produced in the mid metatarsal shaft of all rats was 100% transverse, 73% located at the midshaft. Minimal fracture angulations were recorded (0.48 ± 0.09o; 0.78 ± 0.17o) for anterior-posterior and lateral views respectively. Minimal soft tissue injury was recorded immediately post-surgery, but no infection and the delayed union was observed. Varying degrees of weight-bearing lameness was also recorded but seized at day six onward post-operative. Callus index observed was peaked at week 2 and 3 (2.02 ± 0.1, 1.99 ± 0.13) respectively but declined to 1.10 ± 0.04 at week 7 during the consolidation period. The fracture line disappeared completely at week 7. The histological and radiographic healing scores were (3.5 ± 0.13 and 3.75 ± 0.25) respectively (out of the maximum healing score of 4) at week 7 post-operative. There was a positive correlation between the histological and radiographic healing scores. The metatarsal fracture model is considered to be a suitable model for in vivo study of secondary fracture healing. Doi: 10.28991/SciMedJ-2020-0204-2 Full Text: PDF