Chondrocyte apoptosis enhanced at the growth plate: a physeal response to a diaphyseal fracture.

Post-traumatic overgrowth of growing long bones is a common clinical phenomenon in paediatric traumatology and is the result of an enhanced stimulation of the nearby growth plate after fracture. To date, the exact post-fractural reactions of the growth plate are poorly understood. The aim of this study has been to determine the impact of fracture on the frequency of chondrocyte apoptosis of the growth plate. Rats sustained a mid-diaphyseal closed fracture of the left tibia or were left untreated. All animals were killed 3, 10, 14 or 29 days after trauma. The left and right tibiae were harvested and apoptotic chondrocytes of the proximal tibial growth plate were detected by TUNEL staining. The apoptosis percentage of physeal chondrocytes was statistically compared among fractured bones, intact contra-lateral bones and control bones. The physeal apoptosis rate of the fractured bone was significantly higher than that of the contra-lateral intact bone (valid for all evaluated days) and the control bone (valid from day 10 onwards). Contra-lateral intact tibiae never showed significantly higher apoptosis rates compared with control tibiae. Thus, mid-diaphyseal fracture influences the nearby growth plate by stimulating chondrocyte programmed cell death, which is associated with cartilage resorption and bone replacement. The lack of a significant difference between the intact contra-lateral and the intact control bone suggests that fracture only has a local effect that contributes to the greater apoptosis rate of the adjacent physis.

BMP-6 and BMPR-1a are up-regulated in the growth plate of the fractured tibia.

Bone overgrowth is a known phenomenon occurring after fracture of growing long bones with possible long-term physical consequences for affected children. Here, the physeal expression of bone morphogenetic proteins (BMPs) was investigated in a fracture-animal model to test the hypothesis that a diaphyseal fracture stimulates the physeal expression of these known key regulators of bone formation, thus stimulating bone overgrowth. Sprague-Dawley rats (male, 4 weeks old), were subjected to a unilateral mid-diaphyseal tibial fracture. Kinetic expression of physeal BMP-2, -4, -6, -7, and BMP receptor-1a (BMPR-1a) was analyzed in a monthly period by quantitative real time-polymerase chain reaction and immunohistochemistry. On Days 1, 3, 10, and 14 post-fracture, no changes in physeal BMPs gene-expression were detected. Twenty-nine days post-fracture, when the fracture was consolidated, physeal expression of BMP-6 and BMPR-1a was significantly upregulated in the growth plate of the fractured and contra-lateral intact bone compared to control (p<0.005). This study demonstrates a late role of BMP-6 and BMPR-1a in fracture-induced physeal growth alterations and furthermore, may have discovered the existence of a regulatory "cross-talk" mechanism between the lower limbs whose function could be to limit leg-length-discrepancies following the breakage of growing bones.

Proliferation analysis of the growth plate after diaphyseal midshaft fracture by 5'-bromo-2'-deoxy-uridine.

Both stimulative and inhibitory growth disturbances may occur after a fracture during the growth period. The exact mechanism responsible for stimulative growth disturbances in the immature skeleton is unexplained. It's possible that chondrocyte proliferation leads to overgrowth. This study investigates the effect of a fracture on the proliferation of chondrocytes at the nearby growth plate and its effect on the contra-lateral leg. Fifty-six 1-month-old Sprague-Dawley rats (weight, 100-120 g) were randomised to either an experimental or a control group. A closed mid-diaphyseal tibial fracture was produced in all animals of the experimental group using a standardised technique. On day 3, 10, 14 and 29 of the experiment, the rats were euthanised and their tibial growth plates were subjected to histological analysis. 5'-Bromo-2'-deoxy-uridine labelling was used for the quantitative analysis of chondrocyte proliferation. Safranin O staining provided the histological overview for the subsequent analysis of BrdU-labelling. Immunohistochemical analysis showed increased proliferation of chondrocytes in the growth plates of broken bones during fracture healing. This proliferation peaked on day 3 post-fracture and then reduced gradually until day 29. No increase in the rate of proliferation was observed on the contra-lateral limbs of the animals in the experimental group. Following a diaphyseal fracture of the tibia, the growth plates located next to the fracture react with increased cell proliferation. This proliferation was not observed in the contra-lateral uninjured tibia. This investigation shows that the post-traumatic length discrepancy is a local biological process at the growth plate brought about by the fracture.