See generic requirements in section A.5.1.5.
See generic requirements in section A.5.1.2.
See generic requirements in section A.5.1.3
Proof is required that the validated procedures are performed by the tissue bank staff in a uniform manner in accordance with the approved standard operating procedures.
See generic requirements in section A.184.108.40.206.1
There are various methods used for bone processing by individual tissue banks. From cadaveric donors cancellous and cortico-cancellous, cortical, osteochondral, ligaments and tendons allografts can be processed. Living donor allogeneic and autologous bone allografts are processed in the same manner as allografts. In-between the working steps the bone tissue should be stored at a short time at -40°C, better at -80 °C or kept on ice.
Living donor autogenic chondrocyte culture requires in vitro cell culture procedures.
F.220.127.116.11.1.1. Cancellous and cortico-cancellous bone processing
Cancellous and cortico-cancellous bone grafts might be prepared from epiphysis of distal femur and proximal tibia, proximal and distal epiphysis of humerus and vertebral bodies and iliac crest. Bone tissue should be clined from the remaining soft tissues mechanically. Long bones should be cutted in the border between diaphysis and epiphysis (metaphysis) using different kind of saws (e.g. banding saw, oscilating saw). Final shape of the graft is then achieved.
F.18.104.22.168.1.2. Cortical bone processing
Cortical bone grafts might be prepared from diaphysis of procured long bones: the femur, tibia, fibula, humerus, radius and ulna. Bone tissue should be clined from the remaining soft tissues mechanically. Shape of the graft can be achieved by different kind of saws (see F.22.214.171.124.1.2.)
F.126.96.36.199.1.3. Osteochondral bone processing
Osteochondral bone grafts might be prepared from distal or proximal femur, tibia humerus, radius and ulna. Bone tissue should be clined from the remaining soft tissues mechanically. The shape of the final graft can be achieved by different kind of saws (see F188.8.131.52.1.2.)
F.184.108.40.206.1.4. Ligaments and tendons processing
Achilles and patellar tendons as well as ligaments should be cleaned mechanically.
F.220.127.116.11.1.5. Washing and defatting procedure
Washing and deffating procedure is used to remove cells from cancellous and cortical bone tissue. In the mechanical cleaning process the entire bone marrow is to be removed. Two different methods may be applied: chloroform/methanol (or ethanol) solution (shares of volume 2 : 1) or warm water or saline solutions. In-between the steps of that procedure the rinsing solution should be exchanged. An optical control of the bone as regards structural integrity should be made.
F.18.104.22.168.1.5.1. Defatting procedure
Chloroform/methanol or chloroform/ethanol mixture or alcohol alone should cover the bone to be deffated. The procedure should be done using shaking machine for a period of approximately two hours. In the next step the bone will be rinsed four times in methanol (or ethanol) to remove the chloroform effectively. Finally a rinsing with water or saline solution should be made.
F.22.214.171.124.1.5.2. Washing procedure
Alternatively the removal of cells might be done with a sharp jet or shaking machine with water or saline solution. Warm water in temperature 30oC might be used.
The moisture is extracted from the bone by a process of freezing and prevention of ice formation. The purpose is to permit shelf storage at room temperature. The moisture content at the end of freeze-drying should be less than 5%
Grounded bone (standardized diameter: 80-300 micron, 300-425 micron, 425-600 micron, 600-1000 micron) by freezer mill is subsequently demineralised in acid solution for 90 minutes (e.g. 0.5 or 0.6 M HCl). In next step bone is deffated (as described in F. 126.96.36.199.1.5.1.) and alternatively lyophilised (as described in F. 188.8.131.52.1.6.) or freezed. The amount of calcium (usually less than 10%) content should be calculated (e.g. percent of calcium of bone matrix dry weight).
F.184.108.40.206.1.8. Autologus chondrocyte culture
Samples of articular cartilage should be submitted to enzyme digestion process to isolate cells. After isolation, cells are cultured in the incubator at standard conditions (temperature -37oC, CO2 – 5%, humidity - 95%) in culture flasks with medium (e.g. HAMF12 or DMEM, penicillin/streptomycin 1% and fetal bovine serum 10%) for period of 4-6 weeks. The medium should be changed twice or three times per week. Total cells and percentage of viable cells should be counted after cell isolation and at the end of cultures (e.g. trypan blue in a hemocytometric chamber or in a cell counter). There should be calculated number of viable cell and the average number of cells (cell density). Morphological analyses of cells should be routinely performed using microscope. After enzymatic digestion cultured chondrocytes might be transferred into three dimensional scaffolds (e.g. fibrin glue, aginate rings).
For sterilisation and decontamination wide range of procedures can be used. Sterilisation procedure should assure that none of the viable organisms will be present in the sample after sterilisation. The term Sterility Assurance Level was introduced as the expected probability of a surviving micro-organism on an individual product unit after exposure to a valid sterilisation process. SAL10-6 was established for product with direct contact to human tissues and means, that there is probability of survival of 1 microorganism of 1 mln present in the product.
1. Radiation sterilisation; Both gamma rays and accelerated electron beam might be used for sterilisation process. The technique (irradiation dose, temperature of irradiation) must be validated, considering the initial contamination (bioburden) and other factors influencing effectiveness if radiation-sterilisation (e.g. presence of oxygen, physical state of irradiated graft). No specific dose can therefore be recommended. Doses used for sterilisation ranged from 17 to 35 kGy and are established after calculation of initial contamination. The irradiation process must be always documented.
2. Ethylene oxide sterilisation; the graft should be exposed to the quantity of ethylene oxide recommended by the manufacturer. It should be guaranteed that the procedure meets the requirements of temperature, humidity and gas concentration. After a treatment with ethylene oxide, a ventilation process need to be followed, allowing the elimination of residual ethylene oxide and its by-products (e.g. ethylene chlorohydrins and ethylene glycol). For each batch of ethylene oxide, chemicals indicators should be used. It should be demonstrated that sterilization was reached for each batch of tissue. Representative samples of each batch must be tested to detect the presence of oxide residual ethylene or decomposition products due to the toxicity of chemical residues. The tissue treated with ethylene oxide must then be frozen at a minimum of -40 ° C, or be lyophilized and stored at room temperature.
1. Chemical decontamination; there are many chemicals having a decontamination function or an inactivating effect on specific pathogens (e.g. paracetic acid, iodophors, ethanol). The effectiveness of these agents on certain types of tissue must be validated. It is important to mention the chemicals used in the documentation accompanying the graft.
It is also necessary to mention the nature of these chemicals products and the possible presence of traces of these products or decomposition products.
2. Antibiotic decontamination; for decontamination of musculoskeletal tissue antibiotics may be used. The effectiveness of each antibiotic cocktail should be validated and documented. The use of antibiotic decontamination procedure might be the only method microbial inactivation for chondrocyte cell culture.
The quality control tests on musculoskeletal grafts should consider at least the following minimum quality criteria:
a) Morphology and integrity of the musculoskeletal grafts
b) Shape and a size of a graft
c) Moisture residue in lyophilised grafts
d) Calcium content in demineralised bone
e) Sterilisation indicators
f) Number of viable cells and cell density in chondrocyte culture
g) No evidence of microbiological growth or malignant cells
See generic requirements in section A.5.1.4.
See generic requirements in section A.220.127.116.11
Additionally, antimicrobial effects of antibiotics in the culture medium solution should be taken into account while choosing and validating the microbiological test method for chondrocyte culture.
F.18.104.22.168.2.1. Microbiological controls for musculoskeletal tissues
During procurement microbiological samples should be collected to establish initial contamination of tissues to make a decision during quarantine regarding release of procured material for further processing. Those microbiological tests are also important to control procurement procedure. There might be two techniques of sampling for microbiological testing:
- swabs – collection of potential microorganisms from the surface of tissues
- destructive method – a biopsy taken from procured tissues.
During processing microbiological samples should be collected before packaging of a final product. There might be three techniques of sampling for microbiological testing:
- destructive method
- collection of the last portion of the fluid used for washing of tissue graft.
F.22.214.171.124.2.2. Microbiological controls for cultured chondrocytes
During processing in cell culture at least two microbiological tests should be performed. First sampling of the transport medium should be performed parallel to the enzyme digestion process of articular cartilage.
The second microbiological test should be performed in the end of a chondrocyte culture before release for the clinical use.
In the mean time the culture medium should be regularly inspected for cloudiness, which may indicate contamination. If visible cloudiness or decolouration of the culture medium should occur, suitable microbiological testing should be initiated.
See generic requirements in section A.5.1.5.
See generic requirements in section A.6
Additionally, it must be recognized that while maximum storage times are recommended for the various methods of tissue preservation, in most cases it is preferable to transplant tissues before these maximum times are approached in order to optimize surgical outcome. Availability of tissue, clinical requirements and surgical need may determine the storage time for each individual tissue.
Preservation/storage methods for recovered donor musculoskeletal tissue are presented in chapter F.2.3.6.
1. The packaging material should be validated for storage in hypotermic conditions. The integrity of packaging material plays an important role for assuring microbiological safety.
2. Preservation and storage in the freezer
Preservation and storage of musculoskeletal tissues in the freezer at -20oC to -30oC should not exceed 6 months. Using this method, cancellous, cortico-cancellous, cortical, ligaments and tendons allografts can be preserved and stored.
3. Preservation and storage in deep freezer
Preservation and storage of musculoskeletal tissues in the deep-freezer at -60oC to -80oC allows prolong the storage time up to 5 years. Using this method cancellous, cortico-cancellous, cortical, ligaments and tendons allografts can be preserved and stored.
4. Cryopreservation and storage
Cryopreservation is a process where tissues are preserved by cooling to temperatures approx. −196 °C (the boiling point of liquid nitrogen). Process starts with controlled cooling to -80oC Using this method cancellous, cortico-cancellous, cortical, ligaments and tendons allografts can be preserved and stored. This method is suitable for osteochondral and cells preservation and storage. Cryoprotectants (e.g. glycerol, DMSO - dimethyl sulfoxide) to avoid ice crystal formation that destroy cells are added to freezing medium. The storage time in these conditions should not exceed 5 years.
5. Preservation and storage at room temperature
All kinds of lyophilised musculoskeletal allografts might be stored at room temperature. There is no time limit for storage of lyophilised grafts. The only limitation is the integrity of packaging material to assure microbiological safety and reduced moisture.
6. Tissue culture of chondrocytes
Cultured chondrocytes in conditions described in F.126.96.36.199.1.8. might be cryopreserved with presence of cryoprotectant as described in F.188.8.131.52.1.3. and stored at -196oC. Cultured chondrocytes might be transported in frozen state.
Alternatively, cultured chondrocytes (F.184.108.40.206.1.8) prior to transplantation should be transferred into transport medium. During transport temperatures should range between 2oC to 8oC.
See generic requirements in section A.6
See generic requirements in section A.220.127.116.11.
Before tissues are released for transplantation, all tissue bank records concerning donor screening, testing and quality control should be reviewed and found to be complete and accurate. Final release for transplantation is to be performed and signed by the responsible person. The documentation for the release of donor tissue is to demonstrate that all specifications were met, especially that all effective notification forms, medical records, processing, sterilisation and storage records were inspected.
The tissue bank should establish and document their criteria for release of tissue for transplantation, which should include (but is not limited to), the following:
a) Full donor screening for contraindications with normal result.
b) Negative serological or molecular biological diagnosis of the donor.
c) Acceptable microbiological test results.
d) Comparison of recipient’s for autologous use
e) Macroscopic morphology and integrity of a graft.
f)Shape and size of the graft compared with final label.
g) Sterilisation/decontamination protocol and indicator of process if exists.
h) Moisture residue (water content) in lyophilised grafts.
i) Calcium content in demineralised bone.
j) Integrity of packaging material.
See generic requirements in section A.18.104.22.168.
See generic requirements in section A.22.214.171.124.
See generic requirements in section A.126.96.36.199.