ETHANOL FIXATION VS. FRESH FREEZING:
EFFECTS ON TORSIONAL PROPERTIES OF OVINE TIBIAE

H. Reichel (1), M. Fagan (2), A. Turner (3), K. Ohland (2), H. Aberman (2)
(1) Klinik fr Orthop„die, Martin-Luther-Universit„t Halle-Wittenberg, Halle, Germany
(2) Howmedica Worldwide R&D, Howmedica Inc., Rutherford, NJ 07070
(3) Department of Clinical Science, Colorado State University, Fort Collins, CO 80523

Presented at the 20th Annual Meeting of the American Society of Biomechanics
Atlanta, Georgia. October 17-19, 1996

INTRODUCTION

When investigators from several facilities collaborate, specimens may be tested at sites far from where they are harvested. Often, the specimens are fresh frozen and shipped overnight with dry ice. However, when the collaborations are international the specimens may need to be fixed prior to shipping due to international shipping requirements. While it has been shown that freezing has little effect on the mechanical properties of bone (Sedlin et al., 1966), the effect of fixation on the properties of whole cortical bone, is inconclusive (Sedlin et al., 1966; Linde et al., 1993; Hayes et al., 1979). This study was performed to quantify the changes in the torsional properties of ovine tibiae due to fixation in 70% ethanol.

PROCEDURES

Ten pairs of tibiae were harvested from skeletally mature sheep of the same breed, sex and age. One tibia from each pair was placed in 70% ethanol at room temperature for four weeks. The contralateral tibiae were kept fresh frozen for the same time period at -20_C. Prior to testing, the frozen tibiae were thawed to room temperature and all tibiae were dissected free of soft tissue. The tibiae were then trimmed and each end potted in bone cement using a fixture specially designed to maintain alignment. The testing was performed using a MTS 838 Bionix test machine (Figure 1).

Figure 1: Mechanical testing apparatus

Figure 2: Mode of failure for tested specimen A compressive load of 10N was applied and then the vertical displacement was fixed. The tibiae were tested to failure in torsion by rotating the distal end internally at 15_/min. The maximum torque and angle at maximum torque were recorded. The energy of absorption and the torsional stiffness up to 65% of the maximum torque were calculated using the trapezoidal rule and linear regression, respectively. Paired Student's t-tests, for normal distributions, and Wilcoxon's signed-ranks tests, for skewed distributions, were performed to determine significance between groups.

RESULTS

All tibiae failed by spiral fracture in the diaphysis (Figure 2). Normal distributions were found for maximum torque and torsional stiffness, but not for angle at maximum torque or energy of absorption (Table 1). The maximum torque, angle at maximum torque, and energy of absorption for the ethanol-fixed tibiae were significantly larger than those for the fresh frozen tibiae (p<0.001 in all cases). The torsional stiffness for the fresh frozen tibiae were significantly larger than those for the ethanol-fixed tibiae (p<0.0001). Although the ethanol-fixed tibiae were only ~15% less stiff than the fresh frozen tibiae, the ethanol-fixed tibiae absorbed more than twice as much energy.

Table 1: Torsional Properties of Ovine Tibiae

DISCUSSION

While it is preferable to test specimens that are fresh or have been fresh frozen, it is not always possible. Comparison of the torsional properties between ethanol-fixed and fresh frozen specimens indicates that ethanol fixation increases the compliance of the ovine tibia. This may be due to dehydration of the bone with the resultant loss of fluid stiffening (Ochoa et al., 1991), and/or crosslinking of proteins within the bone matrix. Therefore, storage conditions should be taken into consideration when performing mechanical testing of bones. Furthermore, the use of paired contralateral controls is recommended to limit the effect of storage and handling, as well as inter-subject variability, that could confound the results of the study.

REFERENCES

Hayes, W.C. et al. in Skeletal Research, (p. 267), ed DJ Simmons et al., Academic Press, NY, 1979.

Linde F et al. J Biomech, 26, 1249-52, 1993.

Ochoa JA et al. J Biomech Eng, 113, 259-62, 1991.

Sedlin ED et al. Acta Orthop Scand, 37, 29-48, 1966.