**1. Introduction**

90 Current Frontiers in Cryopreservation

International Organization For Standardization. (2010), ISO 10993-5; Biological evaluation of medical devices – Part 5: Tests for cytotoxicity: in vitro methods. Switzerland

THE UNITED STATES PHARMACOPEIAL CONVENTION. The United States

Rockville: The United States Pharmacopeial Convention, Inc., 2010.

Pharmacopeia. USP 33; NF 28.

The use of allogenic tissues is growing in orthopedic practice, as well as the number of studies on methods for processing, sterilization and cryopreservation that interfere as little as possible with the original physiological properties of the tissues (Nutton et al., 1999).

In addition to bone tissue, other tissues of the locomotor system can be captured, processed and stored in tissue banks with the purpose of transplantation. Therefore a strict quality control must be implemented and set after discussions compiled by international organizations such as AATB e EATB.

The rst report of the use of allografts in humans dates back to 1881. The rst tissue bank of bone grafts was created in 1940 in the United States and the initial clinical results were published in 1942 by Inclan, 1942. Since then a series of regulations and studies has emerged relating to the use of grafts in orthopedic practice19. Currently, tendon allografts are used in knee surgeries, in elbow ligament reconstructions and in revisions of the acromioclavicular joint (Costic et al., 2004).

In our country we have few tissue banks. The tissue bank (BTME) from the Institute of Orthopedics and Traumatology (IOT), Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo was the first and is the biggest bank in activity nowadays. It thas been in operation since 1999 and is governed by local legislation (Amatuzzi et al., 2000). With the restructuring of our service in 2005, we initiated a new technique aimed at the provision of the tendon with a well-structured quality program in line with other centers of excellence nationally and internationally. Today our service is provided by a series of tendons (tibial tendon, Achilles, patellar and peroneal) taken from different regions with very specific applications. Thus, we can then follow the technological trend in the use of

<sup>\*</sup> Caio Oliveira D'Elia1, Antônio Guilherme P. Garofo1, Wagner Castropil1, Luiz Augusto U. Santos2, Marco K. Demange2, José Ricardo Pécora2 and Alberto T. Crocci2

*<sup>1</sup>Vita Institute, Brazil* 

*<sup>2</sup>Universidade de São Paulo; Institute of Orthopedics and Traumatology, Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, SP, Brazil* 

Cryopreservation – A Viable Alternative in

Preparation for Use of Allografts in Knee Ligament Reconstruction 93

the mechanical characteristics of the grafts. The period of 40 days chosen in our service for cryopreservation coincides with the period of incubation necessary for microbiological investigations for bacteria and fungi (Vangsness et al., 2003). The deep-freezing process

Many services prefer to carry out the manipulation of tissues under aseptic conditions from acquisition through clinical use, and the samples are discarded when microbiological assays show positive bacterial cultures (20 to 30%) (Zimmerman et al., 1994). Sterilization methods, therefore, are not completely safe. They can alter the biomechanical characteristics of tissues or fail to penetrate tissue layers, resulting in the protection of microorganisms rather than their destruction. Irradiation with gamma rays is the most common method of sterilization (Sterling et al., 1995). However, to achieve safer sterilization in frozen tissues, high-dose irradiation is necessary, which can alter the biomechanical properties of the tissue in a dose-

Doses as low as 2 Mrad resulted in a statistically significant reduction in biomechanical properties, outcomes, or physical examination measures. Rappe et al. (2007) studied the effect of irradiation on clinical outcomes of ACL reconstruction, they found the irradiated group had an unacceptable higher rate of failure (33%) than the non- irradiated group (2.4%). Fideler et al. (1995) found that the dose of 2.5 Mrad, which was a dose commonly used by tissue banks for sterilization, was just bacteriocidal but ineffective in eliminating viruses such as human immunodeficiency virus (HIV) (Sterling et al., 1995). Doses of 3 to 4 Mrad were necessary to inactivate the virus. Grieb et al. also proved that lower levels of radiation may be inadequate to kill hepatitis and HIV viruses, with a dose of 5 Mrad being necessary (Grieb et al., 2006). When dosage is increased, its clinical implications increase correspondingly. We must question the use of gamma irradiation as there are so many

Also, the sterilization effectiveness against viruses is low (Vangsness et al. 2003). Ethylene oxide sterilization requires strict control of the levels of waste gas in contact with the allograft and is no longer used by tissue banks, due to the possibility of toxic effects for the recipient (dissolution of the graft and articular inammatory reactions) (Vangsness et al. 2003). The processing techniques used in the preparation and preservation of grafts have been questioned as potentially altering the initial resistance and mechanical properties of the

Two studies carried out in Brazil address the biomechanical properties of patellar and calcaneus tendons of cadavers with the same preparation method as that used in our study, comparing fresh and cryopreserved allografts (Giovani et al., 2006; Reiff et al., 2007). They found no differences. A study on metric measurements and attachment levels of the medial patellofemoral ligament shows this to be a distinct structure (Zimmerman et al., 1994). Although there have been studies on the biomechanical behavior of tendons, the literature does not address histological changes of tendons cryopreserved at -80ºC under aseptic conditions (Pearsall et al., 2003). During cryopreservation at -80ºC, thedestruction of the allograft enzyme appears to be minimal and at least one enzyme, collagenase, which can destroy the tissue, is inactive (Tomford, 1997). Furthermore, with cryopreservation there is no intracellular free water, which is thought to be necessary for enzymatic activity, bacterial proliferation and lipid oxidation (Galea & Keamey, 2005; Laitinien et al., 2006). Lipid oxidation inside the tissues induces apoptosis and inhibits cell differentiation; such oxidation

enables storage for up to ve years, and this is the method we use in our service19.

dependent manner (Curran et al., 2004; Fideler et al., 1995).

adverse effects and it fails to sterilize the allograft as required.

graft prior to implantation.

tissues already practiced in other global centers of excellence in orthopedics and traumatology.

In our medical service, Vita Institute (private use), and in the Institute of Orthopedics and Traumatology (public and private use), allografts are used mainly in knee surgeries, ACL reconstruction, multiple ligament reconstructions, ligament surgery in skeletally immature patients and with double bundle reconstruction.
