**10. Lyophilization of musculoskeletal tissue**

50 Current Frontiers in Cryopreservation

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

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,

Illustration 09. Processing Team at work.

type of conservation, and bar code.

Illustration 10. Vacuum sealing and labeling.

analysis.

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 lyophilization chamber.

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 temperature at positive levels.

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

<sup>6</sup>European Association of Tissue Banks. Common Standards for Tissues and Cells Banking: Berlin: European Association of Tissue Banks; 2004.

American Association of Tissue Banks. Standards for Tissue Banking. 11th ed. McLean : American Association of Tissue Banks; 2007

<sup>7</sup> Phillips GO, Strong DM, Versen RV, Nather A. Advances in Tissue Banking. Vol. 4. World Scientific . New Jersey, 2000.

Bancroft JD, Stevens A. Theory and practice of histological techniques. Fourth Edition. Churchill Livingstone. United Kingdom, 1999.

Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 53

The result of the lyophilization is a dry tissue, conservable at room temperature and that should receive final sterilization by radiation. The lyophilized tissue radiation process is carried out in a Multi-purpose Irradiator, with gamma radiation from sources of 60CO. The appropriate radiation dose is 25 kGy, with a dose rate of approximately 7 kGy/h. In order to avoid temperature variation, the samples are radiated in the presence of cooling elements, keeping the temperature

**Time Interval Process Indicators Sample** 

mBar

mBar

mBar

mBar

mBar

mBar

**Phase 02**  RM: 2.32%

**Phase 01**  RM: 6.26%

Temp. Chamber: – 40 ◦C – 80 ◦C

Chamber Vacuum : 8 x 10 -3

Condenser Vacuum : 2 x 10 -3

Chamber Vacuum: 8 x 10 -3

Condenser Vacuum : 1 x 10 -3

**Analysis Final Moisture of** 

**Analysis Temperature: 105◦C**

Chamber Vacuum: 7 x 10 -3

Condenser Vacuum: 1 x 10 -3

**Analysis Final Humidity of** 

Analysis Temperature: 105◦C

Initial Weight: 0.732g Final Weight: 0.715g Solid: 97.68%

Initial Weight: 0.527g Final Weight: 0.494g Solid: 93.74%

**Temperature** 

– 65 ◦C

– 40 ◦C

+ 5 ◦C

between 4 and 8 °C. Red Perspex dosimeters are used for dose control.

**10 minutes** Vacuum Chamber Temp.: – 30 ◦C

**6 hours** Primary Drying Chamber Temp.: – 30 ◦C

**4 hours** Secondary Drying Chamber Temp.: up to + 5 ◦C

Table 1. Primary and Secondary Drying Process

**2 hours** Pre-freezing of the

lyophilization chamber

Illustration 11. Lyophilization chamber: Labconco® equipment from the Tissue Bank of Hospital das Clínicas – Universidade de São Paulo.

Illustrations 12,13. Residual Moisture Analyzer: Ohaus® equipment belonging to the Tissue Bank of Hospital das Clínicas – Universidade de São Paulo.

Illustration 11. Lyophilization chamber: Labconco® equipment from the Tissue Bank of

Bank of Hospital das Clínicas – Universidade de São Paulo.

Illustrations 12,13. Residual Moisture Analyzer: Ohaus® equipment belonging to the Tissue

Hospital das Clínicas – Universidade de São Paulo.

The result of the lyophilization is a dry tissue, conservable at room temperature and that should receive final sterilization by radiation. The lyophilized tissue radiation process is carried out in a Multi-purpose Irradiator, with gamma radiation from sources of 60CO. The appropriate radiation dose is 25 kGy, with a dose rate of approximately 7 kGy/h. In order to avoid temperature variation, the samples are radiated in the presence of cooling elements, keeping the temperature between 4 and 8 °C. Red Perspex dosimeters are used for dose control.


Table 1. Primary and Secondary Drying Process

Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 55

As soon as the quality criteria have been evaluated and approved, the tissues are made

The tissues are distributed to the various specialties (Hip, Knee, Shoulder, Tumors and

The transplanter (physician or dentist) places the order for the tissue through a discussion of cases and by sending a specific form. The tissue reservation takes into account the demand for each type of transplant, waiting list and stock. The waiting list for transplantations performed within the Unified Health System - SUS complies with the prevailing legislation and today is organized and managed by the musculoskeletal tissue banks themselves, observing an order by date of inclusion. Urgent cases appointed by the medical team, such as malignant tumors and situations with a risk of severe complications, are communicated to the musculoskeletal tissue bank through an Emergency Form, for immediate response.

In dentistry, transplants have evolved differently and their distribution features some

Today it can be seen that tissue transplantations in general are on the rise. Events such as officialization in the legislation and the creation of public promotion policies corroborate this evolution. Considering bone transplants alone, 30x growth has been observed in the last 5 years (Brazilian Transplantation Register, 2010), a fact motivated by the start of large-scale distribution of tissues for dental surgery. Although statistics show the number of tissues to be growing, the quantity of donors is still a concern. The vast majority of donors in Brazil are still for the removal of perfused solid organs (heart, kidney, liver, etc. A minority (6% on an average) accept the donation of musculoskeletal tissues. Of these, just 8% on average, become effective donors and the rest are discarded due to the presence of exclusion criteria

Dentistry) according to the availability of and requests for grafts.

**12. Global data on tissue transplantation activities in Brazil** 

such as infections, blood transfusion, and inadequate profile (**Graph 1**).

Graph 1. Reasons for refusals of bone donors between 2006-2010.

(Source: File BTME-HC-USP)

particularities that will be described further on.

available for use.
