**2. Importance of allografts in knee ligament reconstructions**

Grafts are used in various procedures in different branches of orthopedics, including ligament reconstruction. The literature shows the importance of the use of allografts in knee surgery, especially in revision surgeries, multiple ligament reconstructions and, more recently, surgery for primary reconstruction of the anterior cruciate ligament (ACL) in active patients and in those aged over 40 years (Marrale et al., 2007; Sherman & Banffy, 2004). There have been at least 11 clinical studies comparing the use of auto and allografts in the reconstruction of the ACL (Chang et al., 2003; Marrale et al., 2007). Most of these show little difference between the two techniques with respect to long-term results. However, there has been few prospective randomized studies , and the comparison methods (scores), types of graft, as well as methods of preparing and xing the graft are highly variable.

Furthermore, most studies use the patellar tendon graft; therefore, it may not be possible to generalize the conclusions of these studies to the exor tendon. Some authors, such as Lawhorn and Howell, suggest the use of allografts without a bone plug because of the potential for slower incorporation of the bone due to immunogenicity and smaller crosssectional area of transplants with bone plugs (Lawhorn & Howell, 2003). Recently, Sun et al. (2011) published a prospective randomized study comparing non-irradiated allograft with flexors autograft showing similar results between groups in terms of subjective clinical scores, goals, rate of return to sports and incidence of complications (Sun et al., 2011).

However, compared with autologous transplants, allografts do have some advantages. For example, they do not increase morbidity for the donor, they require a shorter surgery time, and they are available without restriction on size and morphology. In ligament reconstruction surgeries, the possibility exists of an immune response from the recipient tunnel enlargement, and delayed incorporation of the allograft (Marrale et al., 2007). The risk of disease transmission and the potential for immunogenicity are the major disadvantages of allografts, but these complications can be controlled (Albert et al., 2006; Barrios et al., 1994, Urabe et al., 2007).

#### **3. Controversy of methods of preparation of allografts**

The increased frequency of the use of allografts in traumato-orthopedics requires the adoption of storage techniques that interfere as little as possible in the quality of the parts (Vangsness et al., 2003). Allografts can be stored in different ways; they can be chilled in residential mechanical freezers at temperatures of + 2ºC to - 4ºC for up to ve days. In freezers with temperatures of -20ºC to -40ºC, they can be stored for up to six months14. At these temperatures, the enzymes present in the tissue are still active and can destroy the tissue. Therefore, storage periods of longer than a few months are not recommended. The methods of sterilization used at low temperatures are effective against fungi and do not seem to change

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

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

Grafts are used in various procedures in different branches of orthopedics, including ligament reconstruction. The literature shows the importance of the use of allografts in knee surgery, especially in revision surgeries, multiple ligament reconstructions and, more recently, surgery for primary reconstruction of the anterior cruciate ligament (ACL) in active patients and in those aged over 40 years (Marrale et al., 2007; Sherman & Banffy, 2004). There have been at least 11 clinical studies comparing the use of auto and allografts in the reconstruction of the ACL (Chang et al., 2003; Marrale et al., 2007). Most of these show little difference between the two techniques with respect to long-term results. However, there has been few prospective randomized studies , and the comparison methods (scores), types of

Furthermore, most studies use the patellar tendon graft; therefore, it may not be possible to generalize the conclusions of these studies to the exor tendon. Some authors, such as Lawhorn and Howell, suggest the use of allografts without a bone plug because of the potential for slower incorporation of the bone due to immunogenicity and smaller crosssectional area of transplants with bone plugs (Lawhorn & Howell, 2003). Recently, Sun et al. (2011) published a prospective randomized study comparing non-irradiated allograft with flexors autograft showing similar results between groups in terms of subjective clinical scores, goals, rate of return to sports and incidence of complications (Sun et al., 2011).

However, compared with autologous transplants, allografts do have some advantages. For example, they do not increase morbidity for the donor, they require a shorter surgery time, and they are available without restriction on size and morphology. In ligament reconstruction surgeries, the possibility exists of an immune response from the recipient tunnel enlargement, and delayed incorporation of the allograft (Marrale et al., 2007). The risk of disease transmission and the potential for immunogenicity are the major disadvantages of allografts, but these complications can be controlled (Albert et al., 2006;

The increased frequency of the use of allografts in traumato-orthopedics requires the adoption of storage techniques that interfere as little as possible in the quality of the parts (Vangsness et al., 2003). Allografts can be stored in different ways; they can be chilled in residential mechanical freezers at temperatures of + 2ºC to - 4ºC for up to ve days. In freezers with temperatures of -20ºC to -40ºC, they can be stored for up to six months14. At these temperatures, the enzymes present in the tissue are still active and can destroy the tissue. Therefore, storage periods of longer than a few months are not recommended. The methods of sterilization used at low temperatures are effective against fungi and do not seem to change

traumatology.

patients and with double bundle reconstruction.

Barrios et al., 1994, Urabe et al., 2007).

**3. Controversy of methods of preparation of allografts** 

**2. Importance of allografts in knee ligament reconstructions** 

graft, as well as methods of preparing and xing the graft are highly variable.

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

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

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

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 graft prior to implantation.

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

Cryopreservation – A Viable Alternative in

donor does not occur (Figures 1 and 2).

Preparation for Use of Allografts in Knee Ligament Reconstruction 95

A very important step of the process of capture is the reconstruction of the donor and for this matter we use prosthesis specially designed using plaster, wire suture, gauze. This reconstruction is done rigorously and is characterized as the most laborious phase of the procedure. All anatomical parameters are respected, and therefore the deformation of the

Fig. 1. Pre-operative preparation of the potential musculoskeletal tissues doner.

Fig. 2. Tissue removal: bone and tendon dissecation under asseptic conditions.

At the end of the uptake, the tissues are sent to BTME chilled in coolers with temperature monitoring throughout the period of transportation. The processing step is preceded by a

**4.2 Processing and cryopreservation of musculoskeletal tissues** 

can be minimized or avoided with cryopreservation at temperatures of at least -70º C (Laitinien et al., 2006). The literature refers to histological changes due to cryopreservation only in cartilage (one of the most commonly used grafts in surgical practice), concluding that during freezing, the vitality of the cells is threatened ( Schchar & McGann, 1986). Other injuries may also occur, such as the formation of extracellular ice crystals, intracellular ice nucleation, collapse of the matrix, and breakage of intercellular bridges.

In our study, the histological study of one tendon (not cartilage) was carried out, and none of these histological phenomena were observed with cryopreservation at -80ºC. Freezing with liquid nitrogen at -179ºC has also been used as a storage method with similar results but higher cost (Zimmerman et al., 1994). Another widespread storage method is lyophilization. Cryopreservation and lyophilization have been related to a reduction in allograft antigenicity (Jackson et al., 1990). The use of chilled saline solution is not a guaranteed method because the stock can only be kept safely for short periods (Zimmerman et al., 1994). Treatment with paraformaldehyde and fixation with glutaraldehyde are no longer recommended because of the toxicity of these solutions to the recipient tissue.

We recently published a study in which we proved the histological properties of the flexor tendons of the knee from cadavers subjected to cryopreservation and experience with the use of allografts of the Knee Group from IOT (Bitar et al., 2010; Damasceno et al., 2009).
