**4. Final considerations**

86 Current Frontiers in Cryopreservation

Fig. 6. Samples of packaging positioned from 0 to 5 for EtO penetration analysis.

Fig. 7 and 8. Performance of the sterility test by direct incubation in samples submitted to

ethylene oxide.

Analyses of the packaging used in this study demonstrated that it is a good option for cryopreservation of tissues at a temperature of – 80ºC.

Our experience with assays to validate coextruded polyethylene and polyamide plastic film shows that the mechanical properties of this material are not altered by cryopreservation and sterilization. Penetration resistance of the thermal seal remained unaltered after all the processes carried out in a tissue bank, such as sterilization and cryopreservation.

We found a loss of barrier due to increased oxygen permeability of around 10% after sterilization and cryopreservation, which can be explained by the humidification of the polyamide. However, this slight alteration in oxygen permeability does not compromise the inner vacuum of the packaging, and does not place at risk the tissue packaged in it.

In relation to total migration, we did not observe any alterations in the assays, i.e. once again, sterilization and cryopreservation did not lead to monomer migration at levels above

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues 89

BRASIL. Ministério da Saúde – Agência Nacional de Vigilância Sanitária. Resolução - RDC

Cory,AH; Owen,TC; Barltrop,JA; Cory, JG. (1991). Use of an aqueous soluble

European Association of Tissue Banks. Common Standards for Tissues and Cells Banking:

EUROPEAN COMMITEE FOR STANDARDIZATION - CEN/TS 13130-16: materials and

EUROPEAN COMMITEE FOR STANDARDIZATION - CEN/TS 13130-21: materials and

EUROPEAN COMMITEE FOR STANDARDIZATION - CEN/TS 13130-26: materials and

EUROPEAN COMMITTEE FOR STANDARDIZATION. EN 1186-1: materials and articles in

EUROPEAN COMMITTEE FOR STANDARDIZATION. EN 1186-14: materials and articles

EUROPEAN COMMITTEE FOR STANDARDIZATION. EN 1186-3: materials and articles in

EUROPEAN PHARMACOPOEIA. 6. ed. Strasbourg, France: European Department for the

Freshney, RI (2000). Culture of animal cells: a manual of basic technique. 4.ed. New York:

GRUPO MERCADO COMÚN. Mercosur//GMC/Res. nº 32/10. Regulamento técnico

using test media iso-octane and 95% ethanol. Brussels, 2002. 20 p.

aqueous food simulants by total immersion. Brussels, 2002. 17 p.

destinados a estar en contato con alimentos. Disponível em:

Berlin: European Association of Tissue Banks; 2004.

test methods for overall migration. Brussels, 2002. 49 p.

1,p.21-34.

p. 75-79.

207-212.

2005. 15p.

2005. 25p.

simulants. February 2005. 17p.

Quality of Medicines, 2008.

Wiley, 577p.

2011

[da] República Federativa do Brasil, Brasília, DF, 20 maio, 1999a. N. 95, Seção

n° 51 de 26 de novembro de 2010. Dispõe sobre migração em materiais, embalagens equipamentos plásticos destinados a entrar em contato com alimentos. Diário Oficial da República Federativa do Brasil, Brasília, DF, 22 dez. 2010. Seção 1, n. 244,

tetrazolium/formazan assay for cell growth assays in culture. Cancer Comm., 3,

articles in contact with foodstuffs - Plastics substances subject to limitation - Part 16: Determination of caprolactam and caprolactam salt in food simulants. February

articles in contact with foodstuffs - Plastics substances subject to limitation - Part 21: Determination of of ethylenediamine and hexamethylenediamine in food

articles in contact with foodstuffs - Plastics substances subject to limitation - Part 26: Determination of 1-octene and tetrahydrofuran in food simulants. February

contact with foodstuffs - plastics. Part 1: guide to the selection of conditions and

in contact with foodstuffs - plastics. Part 14: test methods for "substitute tests" for overall migration from plastics intended to come into contact with fatty foodstuffs

contact with foodstuffs - plastics Part 3: test methods for overall migration into

Mercosur sobre migración en materiales, envases y equipamientos plásticos

<http://www.puntofocal.gov.ar/doc/r\_gmc\_32-10.pdf>. Acesso em: 22 ago.

those required by our legislation. This is also valid for specific migrations of 1-octene, εcaprolactam and hexamethylenediamine.

The application of ethylene oxide is safe for sterilization of this type of packaging, as it results in good penetrability and safe levels of Eto, Etch and Etg residues at the end of the procedure.

In the cytotoxicity test, we observed levels of cell viability of over 90%, therefore they do not become cytotoxic.

Thus, analyses of coextruded plastic polyethylene and polyamide film used in this study proved to be a good option for cryopreservation of tissues at temperatures of –80ºC, even for prolonged periods of 150 days.

#### **5. References**


those required by our legislation. This is also valid for specific migrations of 1-octene, ε-

The application of ethylene oxide is safe for sterilization of this type of packaging, as it results in good penetrability and safe levels of Eto, Etch and Etg residues at the end of the procedure. In the cytotoxicity test, we observed levels of cell viability of over 90%, therefore they do not

Thus, analyses of coextruded plastic polyethylene and polyamide film used in this study proved to be a good option for cryopreservation of tissues at temperatures of –80ºC, even for

American Association of Tissue Banks. Standards for Tissue Banking. 11th ed. Mclean :

ASTM INTERNATIONAL. ASTM F1927-07: standard test method for determination of

ASTM INTERNATIONAL. F 1306-90 (2008) e1: standard test method for slow rate

ASTM INTERNATIONAL. F 88/F 88M-09: standard test method for seal strength of flexible

BRASIL, Leis etc. Decreto n.2268 de 30 de junho de 1997. Dispõe sobre a remoção de órgãos,

BRASIL, Leis etc. Lei n.9434 de 5 de fevereiro de 1997. Dispõe sobre a remoção de órgãos,

BRASIL, Leis etc. Portaria n.1686 de 20 de setembro de 2002. Dispõe sobre a regulamentação

BRASIL, Leis etc. PORTARIA Nº 2.600, DE 21 DE OUTUBRO DE 2009. Aprova o

BRASIL, Leis etc. Resolução n. 220 de 27 de dezembro de 2006. Dispõe sobre o Regulamento

BRASIL. Agência Nacional de Vigilância Sanitária.ANVISA. Resolução nº 105 de 19 de

oxygen gas transmission rate, permeability and permeance at controlled humidity through barrier materials using a coulometric detector. Philadelphia,

penetration resistance of flexible barrier films and laminates. Philadelphia, 1990.

tecidos e partes do corpo humano para fins de transplantes e tratamento. *Diário* 

tecidos e partes do corpo humano para fins de transplantes e tratamento. *Diário* 

para funcionamento de banco de tecidos músculo esqueléticos. *Diário Oficial da* 

Regulamento Técnico do Sistema Nacional de Transplantes. *Diário Oficial da União*,

Técnico para o Funcionamento de Bancos de Tecidos Musculoesqueléticos e de Bancos de Pele de origem humana. *Diário Oficial da União*, Brasília (DF). 2006 29

maio de 1999. Aprova o regulamento técnico sobre disposições gerais para embalagens e quipamentos plásticos em contato com alimentos. Diário Oficial

American Association of Tissue Banks; 2007.

barrier materials. Philadelphia, 2007. 11p.

*União*, Brasília (DF). 2002 24 jul; seção 1:1.

Brasília (DF). 2009 21out.

*Oficial da União*, Brasília (DF). 1997 30 jun; seção 1:1.

*Oficial da União*, Brasília (DF). 1997 5 fev; seção 1:25.

caprolactam and hexamethylenediamine.

become cytotoxic.

**5. References** 

prolonged periods of 150 days.

2007. 6 p.

5p.

dez.

[da] República Federativa do Brasil, Brasília, DF, 20 maio, 1999a. N. 95, Seção 1,p.21-34.


<http://www.puntofocal.gov.ar/doc/r\_gmc\_32-10.pdf>. Acesso em: 22 ago. 2011

**5** 

**Cryopreservation – A Viable Alternative in** 

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.,

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

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

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

Caio Oliveira D'Elia1, Antônio Guilherme P. Garofo1, Wagner Castropil1, Luiz Augusto U. Santos2,

*2Universidade de São Paulo; Institute of Orthopedics and Traumatology, Hospital das Clinicas da Faculdade de* 

Marco K. Demange2, José Ricardo Pécora2 and Alberto T. Crocci2

*Medicina da Universidade de São Paulo, SP, Brazil* 

**1. Introduction** 

organizations such as AATB e EATB.

joint (Costic et al., 2004).

1999).

 \*

*1Vita Institute, Brazil* 

**Preparation for Use of Allografts in Knee** 

**Ligament Reconstruction** 

Alexandre C. Bitar et al.\*

*Vita Institute* 

*Brazil* 

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

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

THE UNITED STATES PHARMACOPEIAL CONVENTION. The United States Pharmacopeia. USP 33; NF 28.
