Repeated freeze–thaw cycles reduce the survival rate of osteocytes in bone-tendon constructs without affecting the mechanical properties of tendons

Abstract

Frozen bone-patellar tendon bone allografts are useful in anterior cruciate ligament reconstruction as the freezing procedure kills tissue cells, thereby reducing immunogenicity of the grafts. However, a small portion of cells in human femoral heads treated by standard bone-bank freezing procedures survive, thus limiting the effectiveness of allografts. Here, we characterized the survival rates and mechanisms of cells isolated from rat bones and tendons that were subjected to freeze–thaw treatments, and evaluated the influence of these treatments on the mechanical properties of tendons. After a single freeze–thaw cycle, most cells isolated from frozen bone appeared morphologically as osteocytes and expressed both osteoblast- and osteocyte-related genes. Transmission electron microscopic observation of frozen cells using freeze-substitution revealed that a small number of osteocytes maintained large nuclei with intact double membranes, indicating that these osteocytes in bone matrix were resistant to ice crystal formation. We found that tendon cells were completely killed by a single freeze–thaw cycle, whereas bone cells exhibited a relatively high survival rate, although survival was significantly reduced after three freeze–thaw cycles. In patella tendons, the ultimate stress, Young’s modulus, and strain at failure showed no significant differences between untreated tendons and those subjected to five freeze–thaw cycles. In conclusion, we identified that cells surviving after freeze–thaw treatment of rat bones were predominantly osteocytes. We propose that repeated freeze–thaw cycles could be applied for processing bone-tendon constructs prior to grafting as the treatment did not affect the mechanical property of tendons and drastically reduced surviving osteocytes, thereby potentially decreasing allograft immunogenecity.

Automated freeze-thaw cycles for decellularization of tendon tissue - a pilot study

“…However, the obtained data suggest a minor importance of the applied cooling rate for decellularization effectiveness in large tendon samples. For further interpretation and especially for a comparison to freeze-thawing-associated effects in other connective tissue types, it should be noted that response to and threshold for cooling rates are cell-specific features [ 31 , 32 ]. Moreover, the disrupting effects of intracellular ice crystals depend on a sufficient duration of thawing [ 30 ].…”

Fabrication and properties of acellular porcine anulus fibrosus for tissue engineering in spine surgery

“…At present, complete decellularization of tissue will require a combination of physical, chemical, and enzymatic approaches [ 6 ]. Compared to a single freeze-thaw treatment, the repeated freeze-thaw of tissues is known to produce most cell destruction and has been used frequently for decellularization of tendinous and ligamentous tissue [ 23 , 26 ]. Following freezing, a subsequent incubation in 0.1% ( w / v ) SDS and nucleases (RNase and DNase) is used to solubilize the cellular remains.…”

Effects of gamma irradiation and repetitive freeze–thaw cycles on the biomechanical properties of human flexor digitorum superficialis tendons

“…The literature lacks documentation concerning the effect of multiple freeze-thaw cycles ( 42 cycles) on tendons and ligaments. In recent studies, repeated freezing-thawing ( Z3 cycles) had histological and biomechanical effects on Achilles tendons in rabbits before allograft tendon transplantation, while Suto et al (2010) showed no significant differences between untreated rat tendons and those subjected to 5 freeze-thaw cycles. In our study, the data support the view that multiple freeze-thaw cycles may adversely affect the biomechanical integrity of fresh allografts.…”