Effects of preservation on the mechanical strength and chemical composition of cortical bone: an experimental study in sheep femora

Abstract

Preservation methods have enabled bone banks to furnish cortical bone grafts to orthopaedic surgeons. However, cortical bone preserved by freezing and autoclaving, may be weakened by these treatments. To test this hypothesis we compared the ultimate tensile strength of freshly harvested sheep femora with that of femora which were frozen at −20°C for 60 days, or autoclaved at 134°C for 8 min.

We measured the collagen and mineral contents (calcium, phosphorus, magnesium) and hydroxyproline of the specimens and tested for changes induced by preservation. Mechanical three point tests showed that frozen femora were significantly stronger than either fresh or autoclaved femora (p<0.05). Frozen specimens also had the highest phosphorus level, indicating these measures are related to strength.

Cortical bone is not significantly weakened by autoclaving or freezing. This result does not imply that preserved grafts are clinically interchangeable with fresh grafts, rather, it suggests that future studies should focus on post surgical issues, such as the rate of remodeling and integration, which may be sensitive to preservation technique.

Introduction

Cortical bone allografts are coming into ever more frequent use. Simple preservation and storage techniques make it possible for tissue banks to deliver cortical bone specimens whenever they are needed, however, the physical changes wrought by preservation procedures are a source of concern. Because cortical allografts are used to restore skeletal integrity, often under weight-bearing conditions, the mechanical strength of the graft is crucial. Grafts weakened by preservation procedures may be liable to fracture.

Changes in the mechanical properties of bone depend on underlying changes in its composition and structural organization [1]. Mineral content and amount of bone determines bone density, which is important for the mechanical properties of bone [2], [3], [4], [5], [6].

Different preservation methods are currently used in bone banks, although their effects on allograft quality are not fully understood. To be broadly applied such methods must be simple, inexpensive, not toxic, sterile, and should effectively preserve the biological and mechanical attributes of the bone.

Bone banks typically store bones in sterile conditions at temperatures below −40°C for periods longer than 6 months or between −18°C and −28°C for periods shorter than 6 months [4]. In bone conserved at −20°C there is no change in physical or mechanical properties but Laforest et al. [7] found deep freezing at –80°C decreases the cortical bone stiffness.

Many authors believe that freezing and freeze-drying are inadequate to prevent infection and insist that bone for allografts should be sterilized by autoclaving, irradiation [3], [8] or chemical treatments [9]. Unfortunately, while the processes of freezing and lyophilization do not alter the mechanical properties of bone, heat and irradiation weaken grafts. Voggenreiter et al. [10] indicated autoclave treatment, at 134°C for 5 min, reduces bone stiffness (28% of the control bone), although treatment for only 3 min maintains acceptable properties. The amount of radiation needed to deactivate HIV in bone is unknown, although it would seem that, at least in vitro, over 3 Mrad are necessary [11], [12]. Some authors report that while such high radiation doses destroy most bacteria and viruses, they may diminish stiffness to compression by up to 50% [12]. Lower radiation doses (0.01–0.05 Mrad) did not appear to reduce the ultimate tensile strength of cortical bone [10].

Heat treatments may also slow the process of integration with host bone [13], although Taguchi et al. [14] found that autoclaved grafts serve as a scaffold for graft incorporation. Boiling bone caused a decrease in stiffness [3] and according to Borchers et al. [15] cortical bone autoclaving and boiling reduces bone strength but freezing and freeze-drying increase bone stiffness [16].

The aim of our study was to determine how conservation methods (freezing and autoclaving) affect the mechanical properties of the bone grafts. We devised a systematic experimental approach using a reproducible model (sheep femora) to test the hypothesis that cortical bone is significantly weakened by freezing and autoclaving. Mechanical strength was measured and followed with extensive chemical analyses of the specimen to determine if any changes in mechanical properties depended on changes in the mineral composition.