Session VI - Basic Science

Sat, 10/9/04 Basic Science, Paper #32, 9:21 am

Traumatic Brain Injury and Enhanced Fracture Healing in a Rat Model

Matthew Boes, MD; Michael Kain, MD; Paul Tornetta, III, MD;
Sanjeev Kakar; Fred Nichols; Dennis Cullinane; Louis Gerstenfeld, PhD; Thomas A. Einhorn, MD;
(all authors a-OTA/Zimmer, Inc. Grant)
Boston University Medical Center, Boston, Massachusetts, USA

Purpose: We tested the hypothesis that rats subjected to traumatic brain injury (TBI) possess a mechanism for enhanced fracture healing that is clinically significant in terms of the biomechanical properties of the callus formed. In addition, we attempted to correlate these biomechanical findings with differential stimulation of osteoblasts in cell culture following exposure to sera from the same head injured animals, as shown in previous studies. No previous study has measured changes in mechanical properties in healing fractures after TBI.

Methods: The protocol was approved by the Institutional Animal Care and Use Committee. Adult male Sprague-Dawley rats (350-375gm) were divided into two groups: treatment (head injury) and control (non-head injury). Animals in both groups received a closed femur fracture that was stabilized with a smooth K-wire similar to a retrograde intramedullary nail. The treatment group received a closed head injury using a well-described model. Four animals from each group were randomly euthanized at two days post-injury with blood samples drawn and harvest of whole brain specimens for histologic analysis and documentation of brain injury. Brain histology findings from the injury group correlated well with findings published in previous reports documenting the effectiveness of the head injury model. The remaining animals in each group were maintained in our animal housing unit for 21 days at which time they were euthanized. Blood samples were drawn from all animals to be used in subsequent in vitro experiments to measure potential mitogenic effects from the sera as well as study the response of various cell culture lines following exposure to the sera. The remaining animals had both fractured and non-fractured femurs removed for biomechanical testing in torsion. These measurements included callus dimensions, ultimate stress (strength) and modulus (stiffness). Total numbers of specimens for biomechanical testing in each group included both femurs from 17 animals in the head injury and fracture (HI&FX) group and 15 animals in the fracture-only (FX-only) group. Data were analyzed using a one-tail Student t-test.

Mitogenic analysis was performed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Established cell lines included C3HT101/2 cells (mouse mesenchymal stem cell phenotype), MC3T3-14 cells (committed mouse osteoblast phenotype) and NIH3T3 (committed mouse fibroblast phenotype). Cells were plated in 24 well plates at 7,500-15,000 cells/well. Cells were treated with media specific to that cell type and supplemented with 5% sera from either HI&FX or FX only groups. Serum was obtained from animals on day 21 after injury. MTT analysis was performed after 24hrs of growth. Experiments were repeated and run with replicates of four for each group. Average values for each group were compared using a two tailed paired students t-test.

Results: Two animals were lost in the HI&FX group due to death from the head trauma at the time of injury. One animal in the FX-only group was excluded due to nonunion at 21 days. There were no non-unions in the HI&FX group. No animals in either group developed signs of infection.

The results of our analysis of callus dimensions revealed a significantly reduced callus size in the HI&FX group. The mean callus diameter for the FX-only group was 7.46 ± 0.74 mm versus 6.66 ± 1.26mm for the HI&FX group (p=0.025).

The results of mechanical testing showed that the calluses of the HI&FX animals experienced a significant increase in stiffness in comparison to FX-only animals (0.313 ± 0.268GPa vs. 0.081 ± 0.108GPa; p=0.006). In addition, although not significant after only 21 days, there was a trend for an increase in torsional strength in the HI&FX group (272.87 ± 101.12Nmm vs. 230.06 ± 80.38Nmm).

Mitogenic analysis revealed a significant increase in proliferation of C3HT10 1/2 cells when exposed to media supplemented with sera from the HI&FX group compared to the FX only group at 24hrs (p=0.00022). The MC3T3 cells and NIH3T3 cells did not have significant difference in proliferation when exposed to the same media.

Significance: These results support those of previous studies that have suggested an increased osteogenic potential as a result of TBI. The findings of a significant increase in callus stiffness associated with a trend for an increase in strength is consistent with the findings of White et al.10 who showed that, in the early stages of normal fracture healing, stiffness is acquired before strength. Thus, there appears to be a quantitative link between TBI and the enhancement of normal fracture healing. These and other associated findings offer new pathways for research to understand mechanisms of fracture healing and how they may be altered to accelerate union and decrease the incidence of delayed or nonunion. The hypothesis that a circulating substance is present in the serum of head-injured patients is closer to validation and merits further study.

This study was funded by an OTA/Zimmer, Inc. Research Grant