**4. Cryopreservation: Our method**

#### **4.1 Tendinous tissue removal**

The attainment of the musculoskeletal tissues has as its source deceased donors with brain death reported by the Committees Intra-hospital - CIHDOTs, Organ Procurement Organizations - OPOS and by the twenty-three central of notification and collection of organs and tissues - CNCDOs, logistically spread throughout country. Notifications for teams pickups are made after the execution of a series of procedures and tests that aim beyond the evidence of brain death, family consent of the donation of organs and tissues.

The donor selection follows a rigorous research with control antigen and antibody serology for HIV, Hepatitis A, B and C, HTLV-1 and 2, syphilis, Chagas disease, toxoplasmosis and cytomegalovirus in addition to testing of last generation for evidencing of DNA (Nucleic Acid Amplification - NAT) for HIV and Hepatitis B and C, required of musculoskeletal tissues. The capture of musculoskeletal tissues (bone and tendons) is performed after the initial screening of donors of multiple organs and tissues (heart, kidney, liver, pancreas, lung, cornea, etc.).

In our specific field, we follow a protocol of evaluation of the donor that counts with a written anamnesis of a term to capture and physical examination. Are excluded donors with orthopedic disorders such as osteoporosis, osteonecrosis, rheumatoid arthritis, lupus erythematosus, malignancy, age that compromises characteristic of tissues, blood transfusion, tattoos or adornments (piercings) within the window period, users of illicit drugs, permanence in endemic areas, generalized or localized infections, fractures, bruises on the limbs which are absorbed in the musculoskeletal tissues or any other situation that would call into doubt the quality of these tissues, as arranged in the existing laws. The tissues removed are immediately packed in triple enclosures, hermetically sealed and sent under refrigeration (- 4 ° C) to the Tissue Bank.

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,

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).

The attainment of the musculoskeletal tissues has as its source deceased donors with brain death reported by the Committees Intra-hospital - CIHDOTs, Organ Procurement Organizations - OPOS and by the twenty-three central of notification and collection of organs and tissues - CNCDOs, logistically spread throughout country. Notifications for teams pickups are made after the execution of a series of procedures and tests that aim beyond the evidence of brain death, family consent of the donation of organs and tissues.

The donor selection follows a rigorous research with control antigen and antibody serology for HIV, Hepatitis A, B and C, HTLV-1 and 2, syphilis, Chagas disease, toxoplasmosis and cytomegalovirus in addition to testing of last generation for evidencing of DNA (Nucleic Acid Amplification - NAT) for HIV and Hepatitis B and C, required of musculoskeletal tissues. The capture of musculoskeletal tissues (bone and tendons) is performed after the initial screening of donors of multiple organs and tissues (heart, kidney, liver, pancreas, lung, cornea, etc.).

In our specific field, we follow a protocol of evaluation of the donor that counts with a written anamnesis of a term to capture and physical examination. Are excluded donors with orthopedic disorders such as osteoporosis, osteonecrosis, rheumatoid arthritis, lupus erythematosus, malignancy, age that compromises characteristic of tissues, blood transfusion, tattoos or adornments (piercings) within the window period, users of illicit drugs, permanence in endemic areas, generalized or localized infections, fractures, bruises on the limbs which are absorbed in the musculoskeletal tissues or any other situation that would call into doubt the quality of these tissues, as arranged in the existing laws. The tissues removed are immediately packed in triple enclosures, hermetically sealed and sent

collapse of the matrix, and breakage of intercellular bridges.

**4. Cryopreservation: Our method** 

under refrigeration (- 4 ° C) to the Tissue Bank.

**4.1 Tendinous tissue removal** 

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 donor does not occur (Figures 1 and 2).

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

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