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Osteogenic Effects of Traumatic Brain Injury on Experimental Fracture-Healing Matthew Boes, Michael Kain, Sanjeev Kakar, Fred Nicholls, Dennis Cullinane, Louis Gerstenfeld, Thomas A. Einhorn and Paul Tornetta, III J. Bone Joint Surg. Am. 88:738-743, 2006. doi:10.2106/JBJS.D.02648

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Osteogenic Effects of Traumatic Brain Injury on Experimental Fracture-Healing BY MATTHEW BOES, MD, MICHAEL KAIN, MD, SANJEEV KAKAR, MD, FRED NICHOLLS, MA, DENNIS CULLINANE, PHD, LOUIS GERSTENFELD, PHD, THOMAS A. EINHORN, MD, AND PAUL TORNETTA III, MD Investigation performed at the Department of Orthopaedics, Boston Medical Center, and the Orthopaedic Research Laboratory, Boston University School of Medicine, Boston, Massachusetts

Background: Heterotopic bone formation has been observed in patients with traumatic brain injury; however, an association between such an injury and enhanced fracture-healing remains unclear. To test the hypothesis that traumatic brain injury causes a systemic response that enhances fracture-healing, we established a reproducible model of traumatic brain injury in association with a standard closed fracture and measured the osteogenic response with an in vitro cell assay and assessed bone-healing with biomechanical testing. Methods: A standard closed femoral fracture was produced in forty-three Sprague-Dawley rats. Twenty-three of the rats were subjected to additional closed head trauma that produced diffuse axonal injury similar to that observed in patients with a traumatic brain injury. Twenty-one days after the procedure, all animals were killed and fracture-healing was assessed by measuring callus size and by mechanical testing. Sera from the animals were used in subsequent in vitro experiments to measure mitogenic effects on established cell lines of committed osteoblasts, fibroblasts, and mesenchymal stem cells. Results: Biomechanical assessment demonstrated that the brain-injury group had increased stiffness (p = 0.02) compared with the fracture-only group. There was no significant difference in torsional strength between the two groups. Cell culture studies showed a significant increase in the proliferative response of mesenchymal stem cells after exposure to sera from the brain-injury group compared with the response after exposure to sera from the fractureonly group (p = 0.0002). This effect was not observed in fibroblasts or committed osteoblasts. Conclusions: These results support data from previous studies that have suggested an increased osteogenic potential and an enhancement of fracture-healing secondary to traumatic brain injury. Our results further suggest that the mechanism for this enhancement is related to the presence of factors in the serum that have a mitogenic effect on undifferentiated mesenchymal stem cells. Clinical Relevance: Fracture-healing may be enhanced by an associated traumatic brain injury. Further understanding of this systemic response could lead to important insights about systemic therapeutic strategies for the enhancement of skeletal repair.

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possible association between traumatic brain injury and increased bone-forming potential has long been recognized. Heterotopic ossification in patients with central nervous system injury has been extensively described1,2. A similar association between traumatic brain injury and enhanced fracture-healing has been observed, although much of the historical evidence of this phenomenon has been anecdotal3. In 1987, two studies demonstrated increased callus formation and a shorter time to union in patients with long-bone fractures and concomitant traumatic brain injury4,5. These results spurred a series of studies over the next several years in which the investigators attempted to identify the mechanism

responsible for accelerated osteogenesis in patients with a traumatic brain injury6-17. Evidence supporting accelerated fracture-healing in patients with a traumatic brain injury is controversial. Garland et al. found no increase in fracture callus and no evidence of shorter healing times in patients with a traumatic brain injury and a fracture of the tibia or femur16,17. Those authors suggested that both periarticular heterotopic ossification and myositis ossificans in patients with a traumatic brain injury are commonly misinterpreted as exuberant fracture callus. Moreover, results of in vitro studies of the effect of serum from patients with a traumatic brain injury on cultured cells have not

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been uniformly conclusive. Although two studies documented activation of osteoblasts on exposure to serum from patients with a brain or spinal cord injury, that finding was not substantiated in a subsequent study6-8. In addition, no substance has been identified as a causative agent, and several authors have believed that the explanation for this phenomenon is probably multifactorial7,9-15. Despite the current lack of conclusive results, a study by Bidner et al. provided strong evidence for the hypothesis that patients with a traumatic brain injury possess a humoral mechanism for enhanced fracture-healing6. Using a welldesigned in vitro protocol, these investigators documented a mitogenic effect of serum from patients with a traumatic brain injury that was specific for cultured osteoblasts. Kurer et al. reported evidence of a similar effect in patients with a spinal cord injury in whom heterotopic ossification had developed8. In addition, multiple reports have documented substantial differences in the constituent composition between serum from patients with a head injury and serum from patients without such an injury9-11,13. However, there is no direct evidence that changes in the serum of patients with a traumatic brain injury lead to clinically important changes in fracture-healing. We sought to test the hypothesis that an association between traumatic brain injury and fracture leads to an osteogenic response and an enhancement of fracture-healing. Wellestablished rat models of both closed femoral fracture and closed head injury were used so that the study groups would differ only in terms of whether or not they had an associated head injury18-20. Mitogenic effects were examined to determine whether selective responses could be measured in mesenchymal stem cells, precommitted osteogenic cells, or fibroblasts, and enhancement of fracture-healing was assessed with biomechanical testing. Materials and Methods ale Sprague-Dawley rats (Harlan Bioproducts for Science, Indianapolis, Indiana), approximately seven to nine months old and weighing an average (and standard deviation) of 449 ± 39 g, were used for all experiments. The animals were individually housed at 22°C with free access to food (standard rat chow) and water on a twelve-hour light-and-dark cycle. Research was conducted in conformity with all Federal and United States Department of Agriculture guidelines as well as with an Institutional Animal Care and Use Committee-approved protocol. In forty-three rats, a closed femoral fracture was produced and then stabilized with a smooth Kirschner wire that was similar to a retrograde intramedullary nail18. The animals were then divided into two groups: one (consisting of twenty animals) in which only the fracture was created (the fracture-only group) and one (consisting of twenty-three animals) in which, in addition to the fracture, a traumatic closed brain injury was produced with an impact acceleration system19,20. This model has been shown to reproducibly impart a diffuse axonal injury similar to that observed in patients with a traumatic brain injury. Postoperatively, a behavior assessment scale was devised in con-

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junction with veterinary supervisors to assess for pain control. Four randomly chosen animals from each group were killed at two days postinjury; blood samples were drawn from them, and whole brain specimens were harvested for histologic analysis and documentation of brain injury. Specimens were prepared and midcoronal sections were cut and stained with hematoxylin and eosin, as previously described, in order to validate the extent of the head injury19,20. The remaining animals in each group were housed for twenty-one days and then killed. Twenty-one days was chosen as the end point for this study on the basis of our previous experience with healing in this rat model. It is the earliest time at which we observed restoration of mechanical properties, and it is also a key transition point between the endochondral and primary bone-formation stages of healing21-24. At this time, blood samples were drawn from all animals. Whole serum was collected by removing the cellular components by centrifugation. Serum was pooled for each group and was stored at −80°C to be used for mitogenic analysis. Surgical Technique Closed, transverse mid-diaphyseal femoral fractures were produced as described by Bonnarens and Einhorn18. Briefly, fractures were generated with the use of a blunt guillotine after stabilization of the femur with an intramedullary pin. General anesthesia was induced with a mixture of isoflurane and oxygen administered with a veterinary inhalation anesthesia machine. The animals were weighed on a digital scale and received an intramedullary injection of 0.2 mL of cefazolin and 0.1 mL of Buprenex (buprenorphine) into the left thigh. The left rear leg was shaved, swabbed with povidone-iodine for disinfection, and draped. A median parapatellar skin incision was made, followed by a median parapatellar incision into the joint capsule extending from the midline through the vastus medialis muscle to the patellar tendon insertion. Slow flexion of the knee and movement of the patella with forceps achieved lateral retraction of the patella on the extended knee. An intercondylar entry point for the insertion of the 0.045inch (1.14-mm) diameter Kirschner wire into the medullary space was made with a handheld drill. The wire was inserted until resistance from the underlying bone of the greater trochanter was met. The wire was then retracted slightly, cut, reinserted, and buried under the knee cartilage surface. The operative site was closed with sutures, and the skin was stapled. The fracture was then produced, and radiographs were made to confirm pin placement and fracture configuration. Fractures that did not occur in the midpart of the diaphysis or that showed excessive comminution were excluded. When the animals were killed, the fractured limb was again radiographed and then was disarticulated at the hip joint. Specimens were harvested and were initially cleaned to remove muscle and soft connective tissue, with care taken not to scratch the bone. At the time of dissection, the callus dimensions were grossly measured in the anteroposterior and mediolateral dimensions with use of digital calipers, and the mean of these

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TABLE I Animal Enrollments and Study Exclusions No. of Animals Brain-Injury Group

Fracture-Only Group

Death

2

0

Histological analysis of brain specimens

4

4

17

15

Biomechanical testing Excluded Total

0 23

1 (nonunion) 20

two measurements was considered to be the overall callus size. Bone specimens were then wrapped in gauze soaked with saline solution and were stored at −20°C. The numbers of animals that were enrolled in the study and the numbers of specimens that were used for the different assessments are presented in Table I. Traumatic Brain Injury The traumatic brain injury was generated with use of an established model19,20. When the animal showed signs of a normal recovery from the creation of the closed femoral shaft fracture, it was reanesthetized with isoflurane. A nickel-plated cap was fitted on the top of the animal’s skull. The animal’s head was then positioned on a foam block under 1.5 m of polyvinyl chloride tubing that was attached to a metal stand. The opening of the tubing was centered over the nickel plate directly above the animal’s head. A 500-g weight was then dropped from the top of the tubing, from a height of exactly 1.5 m. Once impact was made, the foam block and the animal were moved away from the tubing to avoid a rebound impact, thus limiting the injury to a single impaction. Animals were returned to their cages and allowed to recover under observation. Biomechanical Testing Bones were subjected to biomechanical testing to failure with use of a servo-actuated rapid-load torsion testing device at a rate of 10 N/mm/s25. Specimens were thawed just prior to testing and were potted in aluminum blocks at either end in a lead alloy (Cerrobend [Cerro Metal Products, Bellefonte, Pennsylvania]) that melts at a low temperature (70°C). This method allows 1 cm of bone to be exposed (0.5 cm in each direction from the center of the fracture site). The applied moment to and angular deformation of the femora were measured and plotted. Values were obtained for shear modulus (stiffness) and maximum torque (torsional strength). Results were compared between groups with use of a t test of unequal variance and a single-variable analysis of variance. In Vitro Analysis of Serum Serum samples were tested in vitro to evaluate the mitogenic effects of factors within the serum on three different wellestablished murine cell lines. All cell lines were obtained from

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American Type Culture Collection, Rockville, Maryland. The three cell lines were C3H10T1/2 cells, which are representative of a multipotential mesenchymal stem cell line24; MC3T3-14 cells, which are a precommitted osteoblastic cell line25; and NIH3T3 cells, which are a general uncommitted fibroblast cell line26. Mitogenic analysis was performed with use of the commercially available MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide) assay27,28. The MTT assay was selected because of its simplicity and reproducibility for evaluating proliferation of cultured cells and because it does not require the use of radioactivity. The MTT assay measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product released by the mitochondria of viable cells. After incubation of the cells with the MTT reagent, a detergent solution is added to lyse the cells and solubilize the colored crystals. The amount of color produced is directly proportional to the number of viable cells. Cells were plated in twenty-four-well plates at 7.5 × 103 cells per well (for the C3H10T1/2 cells) or 15 × 103 cells per well (for the MC3T3 and NIH3T3 cells). The variation in plating density was intended to adjust for differences in the initial proliferative rate of the cells in order to prevent them from becoming contact-inhibited before the end of the forty-eighthour assay period. For the first twenty-four hours, cells were grown in their respective media as described by American Type Culture Collection29. At the end of this period, the media were changed and normal bovine serum was replaced with 5% rat serum from either the brain-injury or the fracture-only group. After twenty-four hours of growth in the test medium, 50 µL of MTT solute (5 mg/mL in phosphate buffer [SigmaAldrich, St. Louis, Missouri]) was added. At the end of a 2.5hour incubation period, the medium was removed and 500 µL of MTT solution (Sigma-Aldrich) was added to each well of the twenty-four-well plate to dissolve the MTT formazan crystals. Once the crystals were fully dissolved, absorbance was measured with a spectrophotometer at 570 nm with background measurements at 690 nm subtracted out. Experiments were repeated with replicates of four, and values for growth for each cell type exposed to the rat serum were compared between the brain-injury and fracture-only groups for twentyfour hours with use of a two-tailed paired Student t test. Results hile, in our experience, this method of fracture production has been associated with a very low rate of postoperative morbidity and complications, the addition of head trauma increased morbidity substantially (two of the twenty-three animals died). One animal in the fracture-only group was excluded because of a nonunion (a finding of no bone-bridging after pin removal). This rate is similar to that in previous studies24. Interestingly, there were no nonunions in the brain-injury group and no signs of infection in either group. All animals in the braininjury group showed signs of substantial neurologic impairment at the time of injury; these signs consisted of flexed posturing of the forelimbs as well as spastic extension of the hindlimbs and tail, as has been previously observed by other

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TABLE II Biomechanical Properties and Callus Dimensions Measured Twenty-one Days After Fracture Fracture-Only Group* (N = 15)

Brain-Injury Group* (N = 17)

P Value†

Maximum torque (N)

258.4 ± 81

231.4 ± 117

0.472

Stiffness (N/mm)

0.120 ± 0.018

0.306 ± 0.07

0.02

6.66 ± 1.1

0.03

Callus diameter‡ (mm)

7.69 ± 1.7

*The values are given as the mean and standard deviation. †P values were calculated with two separate statistical tests: the t test of unequal variance and a single-variable analysis of variance. The values from the t test are presented. Comparable levels of significance for each data set were obtained with the analysis of variance. ‡Data are shown graphically in Figure 1.

authors19,20. The histological findings in the brain specimens (data not shown) from the brain-injury group correlated well with findings in previous reports documenting the effectiveness of the head injury model19,20. Both groups were able to walk after the injury, and the groups showed no differences in the ability to walk by one day after the injury. Biomechanical Testing Callus dimensions and biomechanical properties were measured to determine if head trauma affected fracture-healing (Table II). The mean fracture callus diameter was significantly reduced (p = 0.030) in the brain-injury group compared with that in the fracture-only group (7.69 mm compared with 6.66 mm; Fig. 1). Mechanical testing after twenty-one days demonstrated that the fracture callus in the brain-injury animals was significantly stiffer than that in the fracture-only animals (0.306 N/ mm compared with 0.120 N/mm; p = 0.02). There was no significant difference in torsional strength between the two groups (258.4 Nm compared with 231.4 Nm; p = 0.472).

Fig. 1-A

In Vitro Analysis of Cell Proliferation The analysis of cell proliferation demonstrated that, compared with the serum from the fracture-only group, the serum from the brain-injury group stimulated the C3H10T1/2 cells to proliferate at a significantly higher level (p = 0.0002), resulting in a 76% increase in cells in the brain-injury group (Table III). In contrast there was no difference in the proliferation of either the MC3T3 cells (p = 0.893) or the NIH3T3 cells (p = 0.779) between the media supplemented with the serum from the brain-injury group and those supplemented with the serum from the fracture-only group. All cell lines demonstrated a continued growth compared with exposure to media only. Discussion he belief that there is an association between traumatic brain injury and fracture repair that enhances fracturehealing remains controversial. Our results support those of previous studies that have suggested that traumatic brain injury decreases fracture-healing time3-6. To the best of our knowledge, we are the first to document alterations in biome-

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Fig. 1-B

Results of the analysis of fracture callus size in the fracture-only (FX) and brain-injury (TBI&FX) groups. A: Mean callus diameters derived from medial-lateral and anterior-posterior measurements. Error bars denote the standard deviation. B: Representative radiographs of femora from the two groups, made twenty-one days following injury.

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TABLE III Absorbance Results of MTT Assay After Twenty-four Hours of Growth* Cell Line

Fracture-Only Group

Brain-Injury Group

P Value

C3H10T1/2

0.135 ± 0.013

0.238 ± 0.010

0.0002

MC3T3

0.153 ± 0.015

0.148 ± 0.041

0.893

NIH3T3

0.097 ± 0.017

0.093 ± 0.038

0.779

*Cell lines were grown in 5% rat serum from the fracture-only or brain-injury group. A spectrophotometer was used to measure absorbance values at 570 nm with a reference value of 690 nm after twenty-four hours of growth. Absorbance is proportional to the number of viable cells.

chanical properties of healing fracture calluses that were associated with a concurrent traumatic head injury. Our data showed a significant increase in callus stiffness. As stiffness is a property of fracture-healing that is acquired earlier than strength, this finding is consistent with the type of enhancement that can be anticipated at the early time-point of twentyone days23. In previous studies detailing the effects of exogenously administered bone morphogenetic protein-2 (BMP-2) to stimulate fracture callus formation in the same animal model, a 34% increase (p = 0.03) in bone stiffness was also shown to precede regains in strength at three weeks30. Other studies relating callus tissue properties to biomechanical properties have also demonstrated that the stiffness of the fracture callus is associated with progressive changes in the tissue’s material properties as hypertrophic cartilage both mineralizes and is replaced with primary bone21,24. The observation of a smaller callus at three weeks in the brain-injury group was unexpected, and without evidence to explain this phenomenon we can only hypothesize about the mechanism behind it. The reduced callus size contradicts the concept that traumatic brain injury increases endochondral ossification and may represent a shift from the recruitment of cells into the endochondral progression to one of more direct bone formation. Alternatively, the smaller calluses in the animals with a traumatic brain injury might suggest that, at twenty-one days, the fractures have already progressed into the remodeling phase of fracture repair. If this was indeed the case, then the lack of significant differences in the strength measurements between these groups may indicate that the synergistic effect between traumatic brain injury and fracture leads to more rapid load-transfer capabilities (as demonstrated by an increase in stiffness) and an acceleration of healing but only a small increase in load-carrying capacity (strength) compared with that in the fracture-only group. In an attempt to expand on the observations made in previous in vitro studies6,8, the current study was designed to identify the cell type involved in the growth-promoting activity of the sera from the rats with a traumatic brain injury. Bidner et al. reported an increased proliferation of osteoblasts in association with traumatic brain injury6. In that study, osteoblasts were represented by primary fetal rat calvarial cells, but a primary cell culture is more accurately a mixture of cell types that includes mature osteoblasts as well as immature stem cells and possibly non-osteogenic cells. We used multiple cell lines in order to determine whether the proliferation was occurring at the stem-cell

level or with more mature cells. The current findings suggest that the proliferative effects of factors in the brain-injury group were specific to mesenchymal stem cells. The putative osteogenic effects of serum from patients with a traumatic brain injury are thought to result from the release of a soluble factor(s) from injured neural tissue or as part of a central nervous system response to brain injury. In this context, two recent studies have shown that neurotrophins and their receptors are both expressed during fracturehealing31 and are found in cartilage and bone cells32. It is also of interest that an association has been noted between nerve growth-factor expression and wound-healing33. Moreover, in recent (unpublished) transcriptional profiling studies of murine fracture-healing carried out in our laboratory, multiple neurotropic factors were shown to be expressed during early time-points after the fracture. The combination of the biomechanical and in vitro data from this and other studies6,8 supports the theory that there is an association between traumatic brain injury and enhancement of fracture-healing. Additional studies to better define this association could lead to therapeutic strategies for the systemic enhancement of skeletal repair. 

Matthew Boes, MD Michael Kain, MD Sanjeev Kakar, MD Paul Tornetta III, MD Department of Orthopaedics, Boston Medical Center, Dowling 2 North, 850 Harrison Avenue, Boston, MA 02118. E-mail address for M. Boes: [email protected]. E-mail address for M. Kain: [email protected]. E-mail address for S. Kakar: [email protected]. E-mail address for P. Tornetta III: [email protected] Fred Nicholls, MA Dennis Cullinane, PhD Louis Gerstenfeld, PhD Orthopaedic Research Laboratory, Boston University School of Medicine, 715 Albany Street, R-205, Boston, MA 02118 Thomas A. Einhorn, MD Department of Orthopaedics, Boston Medical Center, 720 Harrison Avenue, Suite 808, Boston, MA 02118. E-mail address for T. Einhorn: [email protected] In support of their research for or preparation of this manuscript, one or more of the authors received grants or outside funding

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from the Orthopaedic Trauma Association and the Orthopaedic Research and Education Foundation. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable

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or nonprofit organization with which the authors are affiliated or associated.

doi:10.2106/JBJS.D.02648

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1602 COPYRIGHT © 2006

BY

THE JOURNAL

OF

BONE

AND JOINT

SURGERY, INCORPORATED

Errata The Journal publishes corrections when they are of significance to patient care, scientific data or record-keeping, or authorship, whether that error was made by an author, editor, or staff. Errata also appear in the online version and are attached to files downloaded from jbjs.org.

In the article “Osteogenic Effects of Traumatic Brain Injury on Experimental Fracture-Healing” (2006;88:738-43), by Boes et al., the description of the creation of the model should have stated: “In forty-three rats, a closed femoral fracture was produced after stabilization of the femur with a smooth Kirschner wire that was similar to a retrograde intramedullary nail18.” The sentence had erroneously indicated that the stabilization had been performed after the creation of the fracture.

The May Current Concepts Review, “Nontraumatic Osteonecrosis of the Femoral Head: Ten Years Later” (2006;88:1117-32), by Mont et al., contains an error regarding a previously reported dosage. The statement should have read: “Similar findings were recently reported by Lai et al.154, in a study of forty patients with Steinberg Stage-II or III osteonecrosis of the femoral head who were either treated with alendronate (70 mg/wk for twenty-five weeks) or assigned to a control group.”

In the Evidence-Based Orthopaedics analysis of the article entitled "Continuous Passive Motion After Surgery in Infants with Clubfoot Led to Greater Short-Term But Not Long-Term Improvement Relative to Standard Immobilization” by Zeifang et al., with a commentary by Karol (2006;88:1167), the table headings for Cast and CPM were transposed. The table should have read as follows: :

Continual passive motion (CPM) vs standard immobilization (cast) after surgery for clubfoot in infants Mean score (95% confidence interval)* Follow-up

Cast

CPM

P value

6 months

4.2 (3.6 to 4.9)

3.2 (2.8 to 3.7)

0.013

12 months

4.2 (3.5 to 4.8)

3.1 (2.7 to 3.6)

0.009

18 months

4.1 (3.3 to 4.9)

3.2 (2.7 to 3.7)

0.090

48 months

3.9 (2.5 to 5.3)

4.5 (2.7 to 6.2)

0.554

*Dimeglio clubfoot score; range 4 to 16 (worst score).

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