Comparison of bone marrow component removal from processed femoral head bone from living and deceased donors: presence of geodes in living donor bone can prevent maximum removal of marrow components.

NHS Blood and Transplant Tissue and Eye Services banks and issues, cut, shaped and washed bone from deceased donors. The bone is cut/shaped prior to washing and then processed to remove up to 99.9% of blood, bone marrow and associated cells. The processed bone is then sterilised by gamma irradiation with or without a freeze-drying step. Removal of donor blood and bone marrow has been reported to aid incorporation of allograft bone without affecting the biomechanical properties of the bone. However, cut and shaped bone is not suitable for some orthopaedic procedures and some orthopaedic surgeons do not wish to use irradiated bone. Therefore, Tissue and Eye Services have also developed a method for washing intact femoral head bone, from living and deceased donors. We have observed that processing of intact femoral head bone does not always result in removal of 99% (or above) of marrow components and can be as low as 93% removal. We have examined washed femoral head bone and found the presence of internal fluid-filled cysts within subchondral cancellous bone in bone from living donors. The cysts have been identified as geodes and we suggest that these geodes may be responsible for the reduction in bone marrow component removal in living donor bone during processing.

Assessment of a closed wash system developed for processing living donor femoral heads.

NHS Blood and Transplant Tissue and Eye Services (TES) and Scottish National Blood Transfusion Services Tissues and Cells Directorate (TCD) currently bank whole, frozen femoral head bone from living donors who are undergoing primary hip replacement surgery. When required, the bone is issued to a surgeon still frozen on dry ice (- 79 °C). Consequently, the femoral head bone is not processed, is not sterilised and at the time of issue, it contains donor blood, bone marrow and associated cells. We have previously shown that, cut, shaped and washed bone from deceased donors can be processed to remove up to 99.9% of blood, bone marrow and associated cells (Eagle et al. 2015). However, cut and shaped bone is not suitable for some orthopaedic procedures and some orthopaedic surgeons do not wish to use irradiated bone; therefore in this report, a method has been developed in which whole femoral heads can be washed to remove donor blood and bone marrow components. Processing results in excess of 99% bone marrow component removal-soluble protein, haemoglobin and DNA; the procedure is performed inside a closed system, thereby eliminating the need for terminal sterilisation by irradiation. In addition, uniaxial testing demonstrated no difference in compressive strength between washed and unwashed bone. We suggest that this washed bone may be capable of improving incorporation after grafting without disturbing biomechanical properties of the graft.

Validation of cold chain shipping environment for transport of allografts as part of a human tissue bank returns policy.

Human tissue is shipped to surgeons in the UK in either a freeze-dried or frozen state. To ensure quality and safety of the tissue, frozen tissue must be shipped in insulated containers such that tissue is maintained at an appropriate temperature. UK Blood Transfusion Service regulations state "Transportation systems must be validated to show maintenance of the required storage temperature" and also state that frozen, non-cryopreserved tissue "must be transported… at -20 °C or lower" (Guidelines for the Blood Transfusion Services in the United Kingdom, 8th Edn. 2013). To maintain an expiry date for frozen tissue longer than 6 months, the tissue must be maintained at a temperature of -40 °C or below. The objective of this study was to evaluate and validate the capability of a commercially available insulated polystyrene carton (XPL10), packed with dry ice, to maintain tissue temperature below -40 °C. Tissue temperature of a single frozen femoral head or a single frozen Achilles tendon, was recorded over a 4-day period at 37 °C, inside a XPL10 carton with dry ice as refrigerant. The data demonstrate that at 37 °C, the XPL10 carton with 9.5 kg of dry ice maintained femoral head and tendon tissue temperature below -55 °C for at least 48 h; tissue temperature did not rise above -40 °C until at least 70 h. Data also indicated that at a storage temperature lower than 37 °C, tissue temperature was maintained for longer periods.

Production of an osteoinductive demineralised bone matrix powder without the use of organic solvents.

Demineralised bone matrix (DBM) is produced by grinding cortical bone into a powder, sieving the powder to obtain a desired size range and then demineralising the powder using acid. Protocols for the production of DBM powder have been published since 1965 and the powder can be used in lyophilised form or it can be mixed with a carrier to produce a paste or putty. The powder is generally produced from cortical bone which has been processed to remove blood, bone marrow and bone marrow components, including fat. Removal of fat is accomplished by incorporating incubation in an organic solvent, often chloroform, chloroform/methanol or acetone. The use of organic solvents in a clean room environment in a human tissue bank is problematic and involves operator exposure and the potential for the solvent to be trapped in air filters or recirculated throughout the clean room suite. Consequently, in this study, we have developed a cortical bone washing step which removes fat/lipid without the use of an organic solvent. Bone was prepared from six femoral shafts from three donors by dissecting soft tissue and bisecting the shaft, the shafts were then cut into ~9-10 cm lengths. These struts were then taken through a series of hot water washes at 56-59 °C, centrifugation and decontamination steps. Washed cortical struts were then lyophilised before being ground with a compressed air milling machine. The ground bone was sieved, demineralised, freeze-dried and terminally sterilised with a target dose of 25 kGy gamma irradiation. The DBM powder was evaluated for residual calcium content, in vitro cytotoxicity and osteoinductivity by implantation into the muscle of an athymic mouse. Data indicated that in addition to removing in excess of 97% DNA and extractable soluble protein, the washing protocol reduced lipid 10,000-fold. The processed bone was easily ground without clogging the grinder; the sterilised DBM powder was not cytotoxic but was osteoinductive in the animal model. Therefore, we have developed a method of producing osteoinductive DBM without the need to use organic solvents.

Assessment of an improved bone washing protocol for deceased donor human bone.

NHSBT Tissue Services issues bone to surgeons in the UK in two formats, fresh-frozen unprocessed bone from living donors and processed bone from deceased donors. Processed bone may be frozen or freeze dried and all processed bone is currently subjected to a washing protocol to remove blood and bone marrow. In this study we have improved the current bone washing protocol for cancellous bone and assessed the success of the protocol by measuring the removal of the bone marrow components: soluble protein, DNA and haemoglobin at each step in the process, and residual components in the bone at the end of the process. The bone washing protocol is a combination of sonication, warm water washes, centrifugation and chemical (ethanol and hydrogen peroxide) treatments. We report that the bone washing protocol is capable of removing up to 99.85 % soluble protein, 99.95 % DNA and 100 % of haemoglobin from bone. The new bone washing protocol does not render any bone cytotoxic as shown by contact cytotoxicity assays. No microbiological cell growth was detected in any of the wash steps. This process is now in use for processed cancellous bone issued by NHSBT.

The use of a novel bone allograft wash process to generate a biocompatible, mechanically stable and osteoinductive biological scaffold for use in bone tissue engineering.

Fresh-frozen biological allograft remains the most effective substitute for the 'gold standard' autograft, sharing many of its osteogenic properties but, conversely, lacking viable osteogenic cells. Tissue engineering offers the opportunity to improve the osseointegration of this material through the addition of mesenchymal stem cells (MSCs). However, the presence of dead, immunogenic and potentially harmful bone marrow could hinder cell adhesion and differentiation, graft augmentation and incorporation, and wash procedures are therefore being utilized to remove the marrow, thereby improving the material's safety. To this end, we assessed the efficiency of a novel wash technique to produce a biocompatible, biological scaffold void of cellular material that was mechanically stable and had osteoinductive potential. The outcomes of our investigations demonstrated the efficient removal of marrow components (~99.6%), resulting in a biocompatible material with conserved biomechanical stability. Additionally, the scaffold was able to induce osteogenic differentiation of MSCs, with increases in osteogenic gene expression observed following extended culture. This study demonstrates the efficiency of the novel wash process and the potential of the resultant biological material to serve as a scaffold in bone allograft tissue engineering.

Development of a validation protocol to determine the ability of screw-top containers to be watertight and to withstand impact damage from accidental dropping.

Polypropylene screw-top containers are used to collect, transport and store a variety of tissues within tissue banks. These containers are validated for use by tissue banks but no standard validation protocol is used. We present here a protocol for testing screw-top containers for leakage, evaporation and the ability to withstand accidental impact damage. Three different containers were tested, MedFor S072, MedFor S277 and MacoPharma PROT0483. The validation can detect differences between different manufacturer's containers and this protocol will be used in future validations of screw-top containers within National Blood Service Tissue Services.

Investigating the warming and cooling rates of human cadavers by development of a gel-filled model to validate core temperature.

Tissue Services (within NHS Blood and Transplant) plans to bring deceased donors to its state of the art retrieval suite at its new centre in Speke, Liverpool in air-conditioned transport at circa 20 degrees C but without dedicated active cooling. The aim of this study was to determine how quickly a refrigerated body would warm at different ambient temperatures using a gel-filled model. Two models of a human body were prepared consisting of neoprene wetsuits filled with approximately 7 or 18 l of a viscous solution, which once set has similar properties to ballistics gel. This gel consisted of 47.5% distilled water, 47.5% glycerol and 5% agar. Final "dummy" weights were 7.4 and 18.6 kg respectively, representing "virtual" weights of approximately 40 kg and 70 kg. A K-class thermocouple probe was then inserted into a "rectal" position within each model and the models were cooled to a series of different core temperatures: 5 degrees C, 10 degrees C and 15 degrees C and then were placed in an orbital incubator set at 20 degrees C or 30 degrees C ambient temperature. The rate of temperature increase, in the dummy, was measured, until the model's core temperature was close to the ambient temperature. This was done in triplicate for each size model and ambient temperature. Data indicate that increase in core temperature depends on the size of the model and the initial core temperature. For an equivalent donor weight of 70 kg and background temperature of 20 degrees C, core temperature rises from 5 degrees C to 9.2 degrees C; 10 degrees C to 13.3 degrees C and 15 degrees C to 15.5 degrees C after 2 h. The final core temperatures after 2 h are likely to retard bacterial growth, movement or contamination during transport. Cooling rate data indicated that a 70 kg donor equivalent cooled from 37 degrees C to 15 degrees C within 6 h in a cold room at 4 degrees C. This work has shown that a body can be transported without refrigeration and not cause further tissue deterioration as a result.

Validation of radiation dose received by frozen unprocessed and processed bone during terminal sterilisation.

Fresh frozen femoral heads (FH) and frozen processed bone (FP) are widely used as a source of allograft bone. The FP bone and some of the FH are terminally sterilised by the National Blood Service Tissue Services (NSBTS), via application of a minimum 25 kGy gamma radiation dose. To comply with the Guidelines for the Blood Transfusion Services in the United Kingdom (2002), frozen musculoskeletal tissue must be maintained below -40 degrees C during storage and transit. In practice, NBSTS stores bone long-term in -80 degrees C freezers. During transport for irradiation, a temperature of circa -79 degrees C is maintained by packing the bone in dry ice. An evaluation of the radiation dose received by bone has previously been made via dosimeters located within the tissue and dry ice, however, some evidence suggests that low temperature can influence the accuracy of the dosimeter readings. The aim of this study was to determine the actual radiation dose received by FH and FP bone during the irradiation process. This was accomplished by comparing radiation dose readings from dosimeters placed in dry ice with dosimeters placed in a dry ice substitute of similar dimensions and density i.e., polytetrafluoroethylene (PTFE) at ambient temperature. New packing formats were developed for both FH and FP bone such that 15 FH or 3 kg of FP bone could be irradiated in one transport box at any given time in a standardised fashion. The data show that low temperature consistently increased dosimeter readings 10--27%, and that radiation dose always fell within the range of 25--40 kGy (FH=25.1--35.7 kGy; FP bone=25.2--32.4 kGy).