**7. Obtaining musculoskeletal tissues**

44 Current Frontiers in Cryopreservation

Disposal or

Research

Illustration 01. Algorithm of the Musculoskeletal Tissue Donation and Transplantation

Process

The main source of musculoskeletal tissues is the notification of deceased donors to the Transplant Centers, Organ Service Services, and Hospital Transplant Departments. The teams that receive the organs and tissues are only notified after a series of procedures and exams has been carried out to ascertain brain death and obtain the family's consent for the process of organ and tissue donation. Brain death is initially verified by a neurologist, using techniques of physical and imaging (doppler) exams, which are repeated after six hours in the presence of a family member of the potential donor. Once there is no doubt as to the irreversible diagnosis of brain death, the family members are asked whether they would consider donating their loved one's organs. The family interview is done by trained members belonging to an intra-hospital committee, or by an organization that looks for organs. The entire donation process should be recorded and legally signed before the teams are notified to remove each organ (heart, liver, kidney, pancreas, lung, intestine) and tissues (osteochondral and fascial-ligamentous, skin, vessels, cornea, heart valves). Each team should have clearly-defined criteria for selecting, and at the time of notification, accepting or refusing the donor in question.

For donors of musculoskeletal tissue, the selection follows a rigorous control process, with serological tests for antigens and HIV antibodies, Hepatitis A, B and C, HTLV-1 and 2, Syphilis, Chagas disease, Toxoplasmosis and Cytomegalovirus, as well as state-of-the-art tests for evidenciation of D (Nucleic Acid Amplification – NAT) HIV and Hepatitis B and C, bone marrow aspirate smear of the sternum and iliac crest sample, both for histopathological investigation.

Donors with the following criteria were excluded: orthopedic pathologies, such as osteoporosis, osteonecrosis, rheumatoid arthritis, lupus erythematosus, neoplasias, age group that compromises that characteristics of the tissues, blood transfusions, tattoos or piercings within the period of the immunological window, use of illegal drugs, travel to endemic zones, generalized or localized infections, fractures, open sores on the limbs from which the musculoskeletal tissues are to be removed, or any other situation that places in doubt the quality of these tissues, pursuant to the Brazilian legislation.

The whole procedure is carried out in totally antiseptic conditions, just as in surgery. Special gowns made from synthetic material are used, and all the surgical stages of antisepsis are followed.

The removed tissues are immediately packaged in triple packaging, hermetically sealed, and delivered, under refrigeration (-4ºC) to the Tissue Bank.

A very important stage of the capture process is donor reconstruction. The body should be delivered to the family free from any deformation and as close as possible to its appearance before the tissue removal. This is because the fear of deformation has been one of the main

Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 47

Illustration 03. Classified processing room (100 particles/m3 air: Class 100/ISO 5; HEPA

given off by conventional cotton clothing. (**Illustration 04**)

tissue, muscles, fasciae, and fibrotic tissue.

stirring. (**Illustrations 08 and 09**)

enter the environment (e.g. for cleaning and maintenance purposes).

and each procedure requires careful planning. (**Illustrations 05 ,06 and 07**)

The room also has a pass-through anti-chamber, and all the environments have rigorous control of air particles and positive pressure, to ensure the quality of the tissues processed in it. Specific gowning of the professional team is also necessary, using only non-fabric clothing (Spunbond - Meltblown – Spunbond - SMS) to prevent the dispersion of particles

In addition to non-fabric gowning; the team must also adopt certain behaviors. For example, sudden movements, use of cosmetic products and exposure of the skin should be avoided while in this room. Adequate conduct is ensured through special training, not only for the processing team that actually carries out the procedure, but also for other professionals who

A BTME carries out various types of tissue, for use in orthopedic and odontological surgery,

For the processing of fresh, frozen tissues, a process called mechanical processing is carried out, i.e. removal of the adventitious tissues such as the blood, periosteum, subcutaneous

The fragments are then submitted **chemical processing**, where they are immersed in hydrogen peroxide based emulsifying solutions and alcoholic solutions under ultrasound

Filters 99.9 %)

causes of refusals of bone donation by the family. For a perfect reconstruction the professionals use prostheses especially developed for this purpose, plaster, sutured and gauze. (**Illustration 02**). This rigorous reconstruction is the most laborious stage in the removal procedure. Areas possibly visible during the funeral (face, anterior side of arm, anterior side of shoulder, etc.) are not approached. All the anatomical parameters are respected, and therefore donor deformation does not occur.

Illustration 02.Limbs reconstructed with prostheses after tissue removal.

#### **8. Processing the musculoskeletal tissues**

Once the tissues and organs have been obtained, they are delivered to the BTME in portable refrigerators, with temperature monitoring throughout the transport process. The processing stage is preceded by a planning of the activities necessary to accomplish it, such as provision of materials and instruments, summoning the processing team, defining the preparation and dimensions, according to the needs of the service (waiting list) and requests for orthopaedic and odontological surgeons. This stage is done in a special, classified operating theater (class 100 or ISO 5) equipped with a laminar flow module. (Illustration 03). A Class 100 room means it has purity of 100 particules per m3 of air. For the purposes of comparison, an operating theater should have 10,000 particles/m3 of air.

causes of refusals of bone donation by the family. For a perfect reconstruction the professionals use prostheses especially developed for this purpose, plaster, sutured and gauze. (**Illustration 02**). This rigorous reconstruction is the most laborious stage in the removal procedure. Areas possibly visible during the funeral (face, anterior side of arm, anterior side of shoulder, etc.) are not approached. All the anatomical parameters are

respected, and therefore donor deformation does not occur.

Illustration 02.Limbs reconstructed with prostheses after tissue removal.

comparison, an operating theater should have 10,000 particles/m3 of air.

Once the tissues and organs have been obtained, they are delivered to the BTME in portable refrigerators, with temperature monitoring throughout the transport process. The processing stage is preceded by a planning of the activities necessary to accomplish it, such as provision of materials and instruments, summoning the processing team, defining the preparation and dimensions, according to the needs of the service (waiting list) and requests for orthopaedic and odontological surgeons. This stage is done in a special, classified operating theater (class 100 or ISO 5) equipped with a laminar flow module. (Illustration 03). A Class 100 room means it has purity of 100 particules per m3 of air. For the purposes of

**8. Processing the musculoskeletal tissues** 

Illustration 03. Classified processing room (100 particles/m3 air: Class 100/ISO 5; HEPA Filters 99.9 %)

The room also has a pass-through anti-chamber, and all the environments have rigorous control of air particles and positive pressure, to ensure the quality of the tissues processed in it. Specific gowning of the professional team is also necessary, using only non-fabric clothing (Spunbond - Meltblown – Spunbond - SMS) to prevent the dispersion of particles given off by conventional cotton clothing. (**Illustration 04**)

In addition to non-fabric gowning; the team must also adopt certain behaviors. For example, sudden movements, use of cosmetic products and exposure of the skin should be avoided while in this room. Adequate conduct is ensured through special training, not only for the processing team that actually carries out the procedure, but also for other professionals who enter the environment (e.g. for cleaning and maintenance purposes).

A BTME carries out various types of tissue, for use in orthopedic and odontological surgery, and each procedure requires careful planning. (**Illustrations 05 ,06 and 07**)

For the processing of fresh, frozen tissues, a process called mechanical processing is carried out, i.e. removal of the adventitious tissues such as the blood, periosteum, subcutaneous tissue, muscles, fasciae, and fibrotic tissue.

The fragments are then submitted **chemical processing**, where they are immersed in hydrogen peroxide based emulsifying solutions and alcoholic solutions under ultrasound stirring. (**Illustrations 08 and 09**)

Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 49

Illustration 07. Modeling of a jaw bone from a segment of proximal femur.

Illustration 08. Ultrasound washing in emulsifying solution.

Illustration 04. Specific gowning to work in the classified room.

Illustration 05 and 06. Processing of grafts for use in odontological surgery. The photograph on the left shows modeling of maxillary bone defects based on a resin prototype. The one on the right shows fragments of allografts for use in odontological surgery.

Illustration 04. Specific gowning to work in the classified room.

 Illustration 05 and 06. Processing of grafts for use in odontological surgery. The photograph on the left shows modeling of maxillary bone defects based on a resin prototype. The one on

the right shows fragments of allografts for use in odontological surgery.

Illustration 07. Modeling of a jaw bone from a segment of proximal femur.

Illustration 08. Ultrasound washing in emulsifying solution.

Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 51

Once they have been labeled, the tissues are submitted to radiography at the BTME, then

The bones can also be processed in their lyophilized form. The lyophilization process should be validated and be, like all the tissue handling procedures, in conformity with the Manual of Good Manufacturing Practice - GMP and in accordance with international standards6, literature7 and legislation. The procedure involves the use of an automated lyophilization system composed of a Labconco® (**Illustration 11**) freeze drying, or lyophilization chamber with Condensation Chamber/Vacuum. During lyophilization the tissues remain frozen for the prevention of ice crystal liquefaction inside the matrix. The sublimation process should be validated by analyses of the Residual Moisture by automated thermogravimetric method. The lyophilization process is divided into 2 stages: Primary and Secondary Drying. In the primary phase, the largest fraction of water present in the matrix in its solid state (ice crystals) is removed by sublimation induction and gaseous migration. This induction is achieved by the driving force resulting from the difference of pressure gradient between the lyophilization chamber and condenser. The heat generated by this gaseous transportation should be controlled continually by digital sensors strategically positioned inside the

At the end of the sublimation, the aliquot of unfrozen water linked to the organic components of the matrix (proteins) is then removed in the secondary phase, with an increase of pressure in the lyophilization chamber followed by the gradual increase of

The analysis and control of residual moisture are essential to ensure the integrity of the protein matrix. The residual moisture is determined by thermogravimetric method, using an **Ohaus®** (**Illustration 12,13**) moisture analyzer. This method analyzes the initial weight of the sample on precision scales, followed by the promotion of heating with continuous recording of evaporation and weight. The percentage of residual moisture (**RM**), solid mass (**SM**), initial weight (**IW**) and final weight (**FW**) are analyzed in this method. The limit of

6European Association of Tissue Banks. Common Standards for Tissues and Cells Banking: Berlin:

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

Bancroft JD, Stevens A. Theory and practice of histological techniques. Fourth Edition. Churchill

7 Phillips GO, Strong DM, Versen RV, Nather A. Advances in Tissue Banking. Vol. 4. World Scientific .

The musculoskeletal tissue cryopreservation process at - 80◦C is described in chapter 1

**9. Cryopreservation of the musculoskeletal tissues** 

**10. Lyophilization of musculoskeletal tissue** 

**(**VALIDATION OF PRIMARY PACKAGING FOR CRYOPRESERVED

sent for cryopreservation.

lyophilization chamber.

temperature at positive levels.

RM is < **6** % as described by literature (Phillips, 2000).

European Association of Tissue Banks; 2004.

Association of Tissue Banks; 2007

Livingstone. United Kingdom, 1999.

New Jersey, 2000.

MUSCULOSKELETAL TISSUES)

Illustration 09. Processing Team at work.

Immediately afterwards, samples of bone marrow from the long bones and fragments of each tissue submitted to processing are collected from these resulting solutions, and submitted to microbiological processing (general culture, anaerobic and fungal culture) using the direct inoculation technique. Samples are also obtained for histopathological analysis.

Finally, the packaging procedure is begun for all the grafts processed, which are measured (length, height, diameter, weight volume, perimeter), packed in sterile, triple packaging, vacuum sealed, and duly labeled as analysis tissue. (Figure 8). The tissues are labeled with the following information: donor, exams carried out, batch number, item, validity period, type of conservation, and bar code.

Illustration 10. Vacuum sealing and labeling.

Once they have been labeled, the tissues are submitted to radiography at the BTME, then sent for cryopreservation.
