Ralph A. Meyer

Age and ovariectomy impair both the normalization of mechanical properties and the accretion of mineral by the fracture callus in rats.

The impact of age and ovariectomy on the healing of femoral fractures was studied in three groups of female rats at 8, 32 and 50 weeks of age at fracture. In the two older groups, the rats had been subjected to ovariectomy or sham surgery at random at 26 weeks of age. At fracture, all rats received unilateral intramedullary pinning of one femur and a middiaphyseal fracture. Rigidity and breaking load of the femora were evaluated at varying times up to 24 weeks after fracture induction by three‐point bending to failure. Bone mineral density (BMD) was measured by dual‐energy X‐ray absorptiometry. In the youngest group, 8‐week‐old female rats regained normal femoral rigidity and breaking load by 4 weeks after fracture. They exceeded normal contralateral values by 8 weeks after fracture. In the middle group, at 32 weeks of age, fractures were induced, and the femora were harvested at 6 and 12 weeks after fracture. At 6 weeks after fracture there was partial restoration of rigidity and breaking load. At 12 weeks after fracture, only the sham‐operated rats had regained normal biomechanical values in their fractured femora, while the fractured femora of the ovariectomized rats remained significantly lower in both rigidity and breaking load. In contrast, for the oldest group of rats, 50 weeks old at fracture, neither sham‐operated nor ovariectomized rats regained normal rigidity or breaking load in their fractured femora within the 24 weeks in which they were studied. In all fractured bones, there was a significant increase in BMD over the contralateral intact femora due to the increased bone tissue and bone mineral in the fracture callus. Ovariectomy significantly reduced the BMD of the intact femora and also reduced the gain in BMD by the fractured femora. In conclusion, age and ovariectomy significantly impair the process of fracture healing in female rats as judged by measurements of rigidity and breaking load in three‐point bending and by accretion of mineral into the fracture callus.

Comparison of mRNA gene expression by RT-PCR and DNA microarray

Few studies have compared the quantification of mRNA by DNA microarray to the results obtained by reverse transcription PCR (RT-PCR). In this study, mRNA was collected from the healing femoral fracture callus of adult and juvenile rats at various times after fracture. Ten samples were measured by both methods for 26 genes. For RT-PCR, mRNA was reverse transcribed, amplified, electrophoresed, blotted, and probed with 32P-labeled internal oligonucleotides, which were quantified. For DNA microarray, the mRNA was processed to biotin-labeled cRNA, hybridized to 10 Affymetrix Rat U34A microarrays, and quantified. Correlation coefficients (r) for each gene for the agreement between RT-PCR and microarray ranged from -0.48 to +0.93. This variation made the interpretation gene-specific. Genes with moderate expression levels gave the highest r values. Increased numbers of absent calls by the microarray software and increased separation between the location of the PCR primers and the microarray probes both led to reduced agreement. Microarray analysis suggested a floor effect in expression levels measured by RT-PCR for two genes. In conclusion, moderate mRNA expression levels with overlap in the location of PCR primers and microarray probes can yield good agreement between these two methods.

Gene expression in older rats with delayed union of femoral fractures.

Background: Fracture-healing slows with age. While six-week-old rats regain normal bone biomechanics at four weeks after a fracture, one-year-old rats require more than twenty-six weeks. The objective of this study was to examine the possible role of altered mRNA gene expression in this delayed union. Methods: Closed midshaft femoral fractures were created in six-week-old and one-year-old Sprague-Dawley female rats. The animals were killed at zero-time (unfractured) or at 0.4, one, two, three, four, or six weeks after the fracture. mRNA levels were measured by reverse transcription-polymerase chain reaction in the fracture callus for twenty-seven matrix, cytokine, and cytokine-receptor genes for the seven animals per time-point per age-group. Results: The younger rats healed radiographically by four weeks after the fracture, whereas none of the older rats had healed by the sixth week. Despite the difference in healing rates, the levels of mRNA gene expression, in general, followed the same pattern in both age-groups. The mRNA expression levels increased to a peak at one to two weeks after the fracture and then decreased to very low or undetectable levels at four and six weeks after the fracture for both age-groups. Significantly lower levels of mRNA for Indian hedgehog (Ihh) and bone morphogenetic protein-2 (BMP-2) were detected in the fracture calluses of the older rats (p < 0.01 and p < 0.05, respectively). Conclusions: All genes studied were up-regulated by the fracture in both age-groups. Thus, the failure of the older rats to heal promptly was not due to the lack of expression of any of the studied genes. The increase in mRNA for Ihh and BMP-2 in the older rats was smaller than that in the younger rats, which may contribute to slower fracture repair. The return of mRNA gene expression to baseline in the older rats prior to healing may contribute to the delayed union. The slower healing response of the older rats did not stimulate a negative-feedback increase in the mRNA expression of stimulatory cytokines. Clinical Relevance: These data underscore the complexity of regulation of fracture-healing. Cytokine stimulation by the fracture may last for a constant length of time regardless of whether the bone actually unites. Alternatively, one set of cytokines may drive soft callus formation, while another set stimulates bridging callus formation. These data suggest that, if cytokines were administered to enhance bone repair, the age and metabolic status of the patient could influence the optimal timing of the administration.

Lumbar spinal fusion using recombinant human bone morphogenetic protein in the canine. A comparison of three dosages and two carriers.

STUDY DESIGN A randomized, prospective and controlled animal study. OBJECTIVE To evaluate lumbar spinal fusion using recombinant human bone morphogenetic protein 2 in a canine model. SUMMARY OF BACKGROUND DATA Spinal fusion using autogenous bone grafting is associated with donor site morbidity and a nonunion rate of 5% to 35%. The use of recombinant human bone morphogenetic protein 2 as a bone graft substitute would eliminate donor site morbidity and perhaps augment the rate of successful fusion. METHODS Mature beagles underwent bilateral paraspinal exposure at L4-L5, followed by transverse process decortication and randomization into one of six groups using differing doses of recombinant human bone morphogenetic protein 2 implanted using either a Type I collagen carrier or a polylactic acid carrier. Two control groups were used: one group without recombinant human bone morphogenetic protein 2 and another group using autogenous rib graft alone. RESULTS Groups treated with recombinant human bone morphogenetic protein 2 demonstrated complete fusion in all animals. Animals treated with collagen carrier alone (no recombinant human bone morphogenetic protein 2) demonstrated complete absence of fusion. Successful fusion occurred in one of three canines in the autogenous bone graft group. Fusion masses in the recombinant human bone morphogenetic protein 2 treatment groups were significantly larger in size at 3 months than in the autogenous bone graft group. The collagen carrier was more biocompatible and biodegradable because residual polylactic acid carrier was seen with adjacent multinucleated giant cells. There was no evidence of spinal canal or nerve root encroachment in the recombinant human bone morphogenetic protein 2 treatment groups. CONCLUSIONS The use of recombinant human bone morphogenetic protein 2 implanted using a Type I collagen carrier resulted in 100% fusion without adverse effects.

Safety of recombinant human bone morphogenetic protein-2 after spinal laminectomy in the dog.

Study Design. This was a randomized, blinded trial of the safety of the application of recombinant human bone morphogenetic protein (rhBMP)‐2 or autologous bone graft onto a laminectomy defect of the dog in the pres ence or absence of a dural membrane puncture. Objective. To test the safety of rhBMP‐2 in an applica tion in which direct contact of the material with neural tissue occurs. Summary of Background Data. Application of rhBMP‐2 in laboratory animals stimulates local bone for mation to effect spinal fusion and healing of segmental bone defects. The use of rhBMP‐2 as a bone graft substi tute in spinal fusion would eliminate donor site morbidity and may augment the rate of successful fusion. Because rhBMP‐2 may unintentionally come in contact with neural tissue, the consequences of such a safety issue must be addressed in an animal model before human trials. Methods. Twenty skeletally mature beagles under went spinal exposure followed by bilateral laminectomy at L5. In half of the dogs, a puncture wound was made to the dura with the expression of cerebrospinal fluid at the site of the puncture. In randomly selected animals, the exposed dural elements received either autologous bone graft with the bone removed from the laminectomy site or an implant of the rhBMP‐2 device. The animals were observed for 12 weeks with periodic clinical examinations and monthly computed tomographic scans. Results. There was no clinical, radiographic, or histo logic evidence of neurologic abnormalities in these ani mals. The rhBMP‐2 stimulated bone growth in the lami nectomy defect and came into direct contact with the dural membrane. There was no evidence of abnormal mineralization within the thecal sac or in the spinal cord itself. Conclusions. The rhBMP‐2 implant stimulated bone formation in the laminectomy site. Neither autologous bone, rhBMP‐2, nor the dural puncture had deleterious consequences for the animals.

Interobserver Reliability and Intraobserver Reproducibility of the Modified Ficat Classification System of Osteonecrosis of the Femoral Head

Anteroposterior and lateral plain radiographs of 116 osteonecrotic femoral heads were reviewed to assess the interobserver reliability and intraobserver reproducibility of the modified Ficat classification system. The radiographs were reviewed initially and then again six months later by three adult reconstructive surgeons, two general orthopaedic surgeons, two orthopaedic residents, and one musculoskeletal radiologist. All eight observers agreed on the classification of twenty hips (17 per cent) at both the first and the second review of the radiographs. Paired comparisons revealed a mean interobserver kappa reliability coefficient of 0.46 (range, 0.30 to 0.67) for the first review and 0.45 (range, 0.30 to 0.66) for the second. For all observers, the mean rate of perfect agreement between the first and the second review was 68 per cent (range, 56 to 80 per cent). The mean kappa value for intraobserver reproducibility was 0.59 (range, 0.44 [one of the residents] to 0.73 [one of the general orthopaedic surgeons]). No observer or pair of observers had excellent reproducibility or reliability (&kgr; > 0.75). The poor interobserver reliability and fair intraobserver reproducibility diminishes any meaningful comparison of studies in which the modified Ficat classification system has been used and illuminates the need for a more reliable and reproducible classification system.

Lumbar intervertebral disc transfer : a canine study

Study Design. Degenerative lumber disc disease has been implicated as s cause of low back pain. Current treatment options for low back pain involve nonphysiologic fusion of the involved segments and have variable success rates. This is an experimental study of lumbar intervertebral disc transplantation using a canine surgical model. Objectives. This study evaluated the feasibility of lumbar disc transplantation and its effects on disc metabolism and morphology. Method. Eight mature mongrel dogs underwent disc transfer surgeries, in which the L2-L3 and L4-L5 intervertebral discs, with a small segment of adjacent superior and inferior vertebral body, were removed and transposed. The transplanted discs were stabilized by plates or by a flexible cable wire construct using Songer cables (DANEK, Inc. Memphis, TN). Unrestricted activity was allowed postoperatively. At 4 months, the spines were harvested, and the transplanted discs were evaluated biochemically and histologically. Intervening non transplanted discs served as viable controls and thrice-frozen discs served as nonviable controls. Cell viability was assessed by measuring proteoglycan synthesis and DNA content. Results. Proteoglycan synthesis (38S uptake normalized to DNA content) was maintained in transplanted anulus fibroses tissue, but was decreased in nucleus pulposus samples (P < 0.05), DNA content was not altered significantly in the transplanted discs. Histologic analysis of the transplanted discs showed revascularization and remodeling of the bone adjacent to the disc and preservation of the lamellar architecture of the anulus fibrosus. The transplanted nucleus pulposus samples had chondrocyte-like cells present, but the staining characteristics of the nucleus material was variable. The contour of the transplanted disc endplates was irregular in all specimens. Conclusions. The structure and function of autograft intervertebral discs wore maintained after disc transfer surgery; the transplant discs, however, ware not completely normal in either their morphology or their metabolic functioning.

Gene expression during fracture healing in rats comparing intramedullary fixation to plate fixation by DNA microarray.

Objective: This study was designed to compare mRNA gene expression in healing diaphyseal femoral fractures between those injuries treated with intramedullary nails and those treated with internal plate fixation. Design: RNA gene expression was measured at 1 day, 3 days, and 1, 2, 4, and 6 weeks after surgery in the fracture callus of rats randomized to femoral shaft fracture with intramedullary nail fixation, rigid plate fixation, or sham fracture. Setting: AAALAC-accredited vivarium of an independent academic medical center. Animals: Fifty-seven, adult, female, Sprague-Dawley rats at 16 weeks of age. Intervention: Femoral fracture with intramedullary nail fixation, femoral fracture with plate and screw fixation, or sham surgery with no fracture. Main Outcome Measurements: RNA expression for 8700 genes was measured with 19 Affymetrix U34A microarrays. The fracture callus was significantly larger with intramedullary nail fixation than with plate fixation. Most genes responded to fracture with a change in mRNA expression. Most of the responding genes followed the same time course for both fixation methods. This included genes related to growth factors, bone matrix, mast cells, most nerve factors, and hematopoiesis. The intramedullary nail group had significantly greater up-regulation for transcripts related to cartilage, cell division, inflammation, and the acetylcholine receptor. There was significantly greater up-regulation in the plate group for genes related to macrophage activity. Conclusions: There were differentially expressed genes present between the 2 surgical groups that may give insight into the control of fracture repair.

Efficacy of surgical wound drainage in orthopaedic trauma patients: a randomized prospective trial.

OBJECTIVE To study the efficacy of closed suction drainage in clean nonemergent surgical fracture fixation or bone grafting on the extremities or pelvis. DESIGN A prospective randomized trial. SETTING The orthopaedic trauma service of a Level I trauma hospital. PATIENTS Patients were older than age eighteen years and undergoing clean nonemergent surgical fracture fixation or bone grafting procedures on the extremities (excluding hands and feet) or pelvis. INTERVENTION The application of a surgical drain. MAIN OUTCOME MEASUREMENTS Wound drainage, edema, hematoma and erythema, dehiscence, infection, and need for surgery or readmission were followed for six weeks. A univariate analysis with Students t test for continuous variables and chi-squared analysis for all categorical data were used, with a p value of < or = 0.05 considered statistically significant. RESULTS A total of 202 patients were randomized to 102 patients with no drain and 100 patients with a drain. There was no significant difference between the groups with regard to injury severity, systemic disease, age, body weight, physical status, or estimated blood loss. There was no significant difference between the drain and no-drain groups in any of the parameters evaluated. CONCLUSION There is no significant difference between drained and nondrained wounds in clean, nonurgent orthopaedic trauma surgery. It appears that drainage systems can be safely eliminated in this group.

Effect of abnormal mineralization on the mechanical behavior of x-linked hypophosphatemic mice femora

The Hyp mouse is an established animal model of X-linked hypophosphatemia, one of the most common genetic forms of metabolic bone disease in humans. This study describes the first determination of whole bone mechanical behavior in the heterozygous male and female Hyp mouse. Femora from 12-week-old mice were tested in torsion. The contribution of structural and material properties to mechanical behavior was determined by geometrical evaluation prior to testing and by analysis of the diaphyseal mineral after testing. The male and female Hyp femora were found to undergo significantly more angular deformation at failure than the same sex normal femora (82.49 +/- 24.37 vs. 22.63 +/- 8.02 rad/m [corrected] for the females and 128.90 +/- 37.05 vs. 22.79 +/- 7.24 rad/m [corrected] for the males) and to have a significantly lower structural stiffness (0.373 +/- 0.130 x 10(-3) vs. 1.33 +/- 0.380 x 10(-3) [corrected] [N-m/(rad/m)] for the females and 0.167 +/- 0.104 x 10(-3) vs. 1.60 +/- 0.502 x 10(-3) [corrected] [N-m/(rad/m)] for the males). The male Hyp femora had a significantly lower failure torque than male normal femora (1.58 +/- 0.62 x 10(-2) vs. 3.44 +/- 1.57 x 10(-2) N-m). Because the polar movement of inertia, a geometrical property that affects torsional behavior, was not significantly different between the Hyp femora and the same sex normals, differences in mechanical behavior were attributed to material properties.(ABSTRACT TRUNCATED AT 250 WORDS)