A combined physicochemical approach towards human tenocyte phenotype maintenance.

During in vitro culture, bereft of their optimal tissue context, tenocytes lose their phenotype and function. Considering that tenocytes in their native tissue milieu are exposed simultaneously to manifold signals, combination approaches (e.g. growth factor supplementation and mechanical stimulation) are continuously gaining pace to control cell fate during in vitro expansion, albeit with limited success due to the literally infinite number of possible permutations. In this work, we assessed the potential of scalable and potent physicochemical approaches that control cell fate (substrate stiffness, anisotropic surface topography, collagen type I coating) and enhance extracellular matrix deposition (macromolecular crowding) in maintaining human tenocyte phenotype in culture. Cell morphology was primarily responsive to surface topography. The tissue culture plastic induced the largest nuclei area, the lowest aspect ratio, and the highest focal adhesion kinase. Collagen type I coating increased cell number and metabolic activity. Cell viability was not affected by any of the variables assessed. Macromolecular crowding intensely enhanced and accelerated native extracellular matrix deposition, albeit not in an aligned fashion, even on the grooved substrates. Gene analysis at day 14 revealed that the 130 kPa grooved substrate without collagen type I coating and under macromolecular crowding conditions positively regulated human tenocyte phenotype. Collectively, this work illustrates the beneficial effects of combined physicochemical approaches in controlling cell fate during in vitro expansion.

Cost effectiveness of using Faecal Immunochemical Testing (FIT) as an initial diagnostic investigation for patients with lower gastrointestinal symptoms suggestive of malignancy.

BACKGROUND: There has been an increase in the numbers of patients presenting to primary care with suspected colorectal malignancy and subsequently an increase in demand for endoscopy. This study aims to forecast the cost of faecal immunochemical testing (FIT) compared to conventional diagnostic tests as a primary investigation for patients with symptoms suggestive of colorectal malignancy. METHODS: Retrospectively, 1950 patients with symptoms suggestive of colorectal malignancy who were referred through primary care and underwent investigations through standard endoscopic evaluation were included. These patients were used to forecast the cost of faecal immunochemical testing creating theoretical data for sensitivity and specificity. Outcome measures included: the number of investigations under current protocol; cost of current investigations; number of predicted false negatives and false positives and positive/negative predictive values using current sensitivity data for FIT; the cost forecast of using FIT as the primary investigation for colorectal malignancy. RESULTS: Median age was 65 (IQR 47-82) with 43.7% male and 56.3% female. A total of 1950 investigations were carried out with a diagnostic yield of 26 cancers (18 colon, 8 rectal), 138 polyps and 29 high risk adenomas (HGD ± > 10 mm). In total, £713,948 was spent on the investigations. The commonest investigation was colonoscopy totalling £533,169. The total cost per cancer diagnosis was £27,459. Sensitivity (92.1% CI 86.9-95.3) and specificity (85.8% CI 78.3-90.1) for FIT in colorectal cancer was taken from NICE and was costed via the manufacturer(s). The projected total cost of FIT for the same population using a ≥ 4 µg haemoglobin cut off was £415,680 (£15,554 per cancer). The total cost of high-risk polyps using ≥ 4 µg cut off was £404,427 (sensitivity 71.2% CI 60.5-87.2, specificity 79.8%CI 76.1-83.7) or £13,945 per polyp. CONCLUSIONS: FIT is a cheaper and effective alternative test with the potential to replace current expensive methods. The forecast is based on the limited data available for sensitivity/specificity in the current literature. FIT has now been commenced for symptomatic patients in the UK and therefore sensitivity may change in the future.

Investigation of a foodborne outbreak of Shigella sonnei in Ireland and Northern Ireland, December 2016: the benefits of cross-border collaboration and commercial sales data.

OBJECTIVES: To describe a cross-border foodborne outbreak of Shigella sonnei that occurred in Ireland and Northern Ireland (NI) in December 2016 whilst also highlighting the valuable roles of sales data and international collaboration in the investigation and control of this outbreak. STUDY DESIGN: A cross-border outbreak control team was established to investigate the outbreak. METHODS: Epidemiological, microbiological, and environmental investigations were undertaken. Traditional analytical epidemiological studies were not feasible in this investigation. The restaurant chain provided sales data, which allowed assessment of a possible increased risk of illness associated with exposure to a particular type of heated food product (product A). RESULTS: Confirmed cases demonstrated sole trimethoprim resistance: an atypical antibiogram for Shigella isolates in Ireland. Early communication and the sharing of information within the outbreak control team facilitated the early detection of the international dimension of this outbreak. A joint international alert using the European Centre for Disease Control's confidential Epidemic Intelligence Information System for Food- and Waterborne Diseases and Zoonoses (EPIS-FWD) did not reveal further cases outside of the island of Ireland. The outbreak investigation identified that nine of thirteen primary case individuals had consumed product A from one of multiple branches of a restaurant chain located throughout the island of Ireland. Product A was made specifically for this chain in a food production facility in NI. S. sonnei was not detected in food samples from the food production facility. Strong statistical associations were observed between visiting a branch of this restaurant chain between 5 and 9 December 2016 and eating product A and developing shigellosis. CONCLUSIONS: This outbreak investigation highlights the importance of international collaboration in the efficient identification of cross-border foodborne outbreaks and the value of using sales data as the analytical component of such studies.

Ileo-anal pouch excision and permanent ileostomy - Indications and outcomes from a tertiary centre.

PURPOSE: Pouch excision is a major complication of ileoanal pouch surgery. Current practice is for this type of surgery to be performed in a specialist centre. We present a series of patients undergoing pouch excision surgery in a high volume centre in the UK and assess the outcomes in these patients. METHODS: All patients undergoing pouch excision at the Royal Liverpool Hospital between 1995 and 2015 under the care of a single surgeon were included. Demographics and outcomes were taken from patients' notes and a dedicated retrospectively compiled database. RESULTS: 35 patients underwent pouch excision surgery during this period. Around half the patients had their original pouch surgery elsewhere and were referred for management of complications. Median time to pouch excision was 13 years from the original operation. Overall complication rate was 31% with 11% requiring re-intervention post-operatively. There was no mortality in this series. CONCLUSION: Pouch excision is a complex, high-risk procedure that should be carried out in specialist centres. Our series shows that in such settings, good outcomes can be achieved for these patients.

The epidemiology and management of candidemia in Northern Ireland during 2002-2011, including a 12-month enhanced case review.

In Northern Ireland there are concerns about candidaemia, with rates higher than those reported in England and Wales. Our aim was to explore the epidemiology of candidaemia during a 10 year period and the clinical management upon suspicion of cases during a one year enhanced investigation in Northern Ireland.Candidaemia reports to the Public Health Agency were validated during 2002-2011 and used to examine incidence and antifungal sensitivity trends (during 2007-2011). A clinical proforma was used to collate information for all patients with candidaemia in 2011.The majority (96%) of isolates were captured through voluntary laboratory reporting. There was a year-on-year increase in candidaemia from 2002-2011, from 80 to 131 episodes (incidence rate ratio 1.09 95% CI 1.05-1.13). Rates were highest in males under 1 year and over 75 years. 83/98 (85%) of case notes were available from candidaemia patients during 2011. The most prevalent risk factors were patients on total parenteral nutrition (26 people, 31.3%), surgery in the two months prior to the candidaemia (25 people, 30.1%), significant steroid use in the previous 3 months (24 people, 28.9%) and active neoplastic disease (23 people, 27.7%),This study confirmed an increase in candidaemia rates over time, with the observed incidence in 2011 higher than England and Wales. We identified areas for improvement around the clinical management of candidaemia. We recommend raising the awareness of guidelines for fundoscopy, echocardiography and central venous catheter removal.

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 the permeability properties of cryopreservation outer bags used in NHSBT.

This study was carried out to investigate leakage/transport across the bag material of six outer cryopreservation bags in common use within NHS Blood and Transplant. In order to do this two different leak testing procedures; coloured dye and hydrogen tracer gas, were used. The data obtained show that a coloured dye cannot permeate through the materials both at room temperature and following storage at liquid nitrogen temperature (- 196 °C). In addition, when filled with the smallest elemental molecule, hydrogen, in the form of a tracer gas, all of the bags only allowed trace amounts of hydrogen to escape, either through the seal or the bag material. The data indicated that each of the bag materials tested would be capable of preventing bacterial or viral cross-contamination as long as the material remained intact.

In vitro biomechanical and hydrodynamic characterisation of decellularised human pulmonary and aortic roots.

BACKGROUND AND PURPOSE OF THE STUDY: The use of decellularised biological heart valves in the replacement of damaged heart valves offers a promising solution to reduce the degradation issues associated with existing cryopreserved allografts. The purpose of this study was to assess the effect of low concentration sodium dodecyl sulphate decellularisation on the in vitro biomechanical and hydrodynamic properties of cryopreserved human aortic and pulmonary roots. METHOD: The biomechanical and hydrodynamic properties of cryopreserved decellularised human aortic and pulmonary roots were fully characterised and compared to cellular human aortic and pulmonary roots in an unpaired study. Following review of these results, a further study was performed to investigate the influence of a specific processing step during the decellularisation protocol ('scraping') in a paired comparison, and to improve the method of the closed valve competency test by incorporating a more physiological boundary condition. RESULTS: The majority of the biomechanical and hydrodynamic characteristics of the decellularised aortic and pulmonary roots were similar compared to their cellular counterparts. However, several differences were noted, particularly in the functional biomechanical parameters of the pulmonary roots. However, in the subsequent paired comparison of pulmonary roots with and without decellularisation, and when a more appropriate physiological test model was used, the functional biomechanical parameters for the decellularised pulmonary roots were similar to the cellular roots. CONCLUSION: Overall, the results demonstrated that the decellularised roots would be a potential choice for clinical application in heart valve replacement.

Development and characterisation of a large diameter decellularised vascular allograft.

The aims of this study were to develop a biological large diameter vascular graft by decellularisation of native human aorta to remove the immunogenic cells whilst retaining the essential biomechanical, and biochemical properties for the ultimate benefit of patients with infected synthetic grafts. Donor aortas (n = 6) were subjected to an adaptation of a propriety decellularisation process to remove the cells and acellularity assessed by histological analysis and extraction and quantification of total DNA. The biocompatibility of the acellular aortas was determined using standard contact cytotoxicity tests. Collagen and denatured collagen content of aortas was determined and immunohistochemistry was used to determine the presence of specific extracellular matrix proteins. Donor aortas (n = 6) were divided into two, with one half subject to decellularisation and the other half retained as native tissue. The native and decellularised aorta sections were then subject to uniaxial tensile testing to failure [axial and circumferential directions] and suture retention testing. The data was compared using a paired t-test. Histological evaluation showed an absence of cells in the treated aortas and retention of histoarchitecture including elastin content. The decellularised aortas had less than 15 ng mg-1 total DNA per dry weight (mean 94% reduction) and were biocompatible as determined by in vitro contact cytotoxicity tests. There were no gross changes in the histoarchitecture [elastin and collagen matrix] of the acellular aortas compared to native controls. The decellularisation process also reduced calcium deposits within the tissue. The uniaxial tensile and suture retention testing revealed no significant differences in the material properties (p > 0.05) of decellularised aorta. The decellularisation procedure resulted in minimal changes to the biological and biomechanical properties of the donor aortas. Acellular donor aorta has excellent potential for use as a large diameter vascular graft.

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.

The efficacy and sterilisation of human decellularised dermal allografts with combinations of cupric ions and hydrogen peroxide.

Decellularised tissue allografts have been used in reconstructive surgical applications and transplantation for many years. Some of the current methods of sterilisation have a detrimental effect on the tissue graft structure and function. The anti-microbial activity of cupric ions and hydrogen peroxide (H2O2) are well known however their combined application is not currently utilised as a decontamination agent in the tissue banking world sector. The aim of this study was to determine the combined concentrations of copper chloride (CuCl2) and H2O2 that have the optimal bactericidal and sporicidal activity on decellularised (dCELL) human dermis. The first part of this study established the decimal reduction time (D-value) of CuCl2 (0.1 mg/L and 1 mg/L) together with H2O2 (0.01, 0.1, 0.5 and 1%) for Staphylococcus epidermidis, Escherichia coli and Bacillus subtilis spores. The second part of this study identified the most effective CuCl2 and H2O2 concentration that decontaminated dCELL human dermis inoculated with these pathogens. Of all the concentrations tested, 0.1 mg/L CuCl2 in combination with 1% H2O2 had the shortest D-value; S. epidermidis D = 3.15 min, E. coli D = 2.62 min and B. subtilis spores D = 18.05 min. However when adsorbed onto dCELL dermis, S. epidermidis and E. coli were more susceptible to 1 mg/L CuCl2 together with 0.5% H2O2. These studies show promise of CuCl2-H2O2 formulations as potential sterilants for decellularised dermal allografts.

Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells.

The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variability and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmission, we sought to determine if decellularisation and/or γ-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without γ-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, ΔNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and γ-irradiation did not significantly affect the LSC phenotype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p<0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a γ-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer. STATEMENT OF SIGNIFICANCE: Despite its disadvantages, including its biological variability and its ability to transfer disease, human amniotic membrane (HAM) remains the gold standard substrate for limbal stem cell (LSC) culture. To address these disadvantages, we used a decellularised HAM sterilised by gamma-irradiation for LSC culture. We cultured LSCs on fresh HAM, HAM decellularised with NaOH, HAM decellularised with sodium dodecyl sulfate (SDS) and HAM decellularised with SDS and sterilised with gamma-irradiation. We demonstrated that although HAM decellularised with SDS and sterilised with gamma-irradiation is significantly stiffer this does not affect LSC culture growth rate or the phenotype of cultured LSCs. We therefore recommend the use of SDS decellularised gamma-irradiated HAM in future LSC clinical trials.

Pre-hospital assessment with ultrasound in emergencies:implementation in the field / 世界急诊医学杂志（英文）

@#BACKGROUND: Point-of-care ultrasound (US) is a proven diagnostic imaging tool in the emergency department (ED). Modern US devices are now more compact, affordable and portable, which has led to increased usage in austere environments. However, studies supporting the use of US in the prehospital setting are limited. The primary outcome of this pilot study was to determine if paramedics could perform cardiac ultrasound in the field and obtain images that were adequate for interpretation. A secondary outcome was whether paramedics could correctly identify cardiac activity or the lack thereof in cardiac arrest patients. METHODS: We performed a prospective educational study using a convenience sample of professional paramedics without ultrasound experience. Eligible paramedics participated in a 3-hour session on point-of-care US. The paramedics then used US during emergency calls and saved the scans for possible cardiac complaints including: chest pain, dyspnea, loss of consciousness, trauma, or cardiac arrest. RESULTS: Four paramedics from two distinct fire stations enrolled a total of 19 unique patients, of whom 17 were deemed adequate for clinical decision making (89％, 95％CI 67％–99％). Paramedics accurately recorded 17 cases of cardiac activity (100％, 95％CI 84％–100％) and 2 cases of cardiac standstill (100％, 95％CI 22％–100％). CONCLUSION: Our pilot study suggests that with minimal training, paramedics can use US to obtain cardiac images that are adequate for interpretation and diagnose cardiac standstill. Further large-scale clinical trials are needed to determine if prehospital US can be used to guide care for patients with cardiac complaints.

EVALUATION OF DMSO TRANSPORT IN HUMAN ARTICULAR CARTILAGE: VEHICLE SOLUTIONS AND EFFECTS ON CELL FUNCTION.

Osteochondral allografting techniques are limited by the availability of suitable donor tissue; there is an urgent need for effective cryopreservation. A fundamental requirement is the need to establish initial conditions of exposure to cryoprotectant that the chondrocytes will tolerate and that load the tissue with an adequate concentration of cryoprotectant. Three vehicle solutions to transport DMSO into the tissue were studied. Knee joints were obtained from deceased donors with appropriate consent. Whole condyles were treated with 20% w/w DMSO in each of three vehicle solutions and chondrocyte function and tissue CPA content measured. The results showed that exposure to 20% DMSO in each vehicle solution for 2 hours at 0 degrees C was tolerated without loss of GAG synthetic activity. It was observed that penetration of DMSO increased little after 1 hour of CPA exposure at 0 degrees C but the final tissue concentration of CPA was markedly lower than that in the medium.

A liquidus tracking approach to the cryopreservation of human cartilage allografts.

In the "liquidus tracking" (LT) approach to cryopreservation both the temperature and the concentration of cryoprotectant (CPA) are controlled such that solution composition "tracks" the liquidus (melting point) line for that system. Ice crystal formation is prevented but the tissue is not exposed to CPA concentrations exceeding those experienced by cells during conventional cryopreservation. This approach is particularly appropriate for articular cartilage because chondrocytes in situ are exquisitely susceptible to damage by the crystallisation of ice. This project aimed to develop a suitable process for tissue to be used in the surgical repair of damaged human knee joints. A high proportion of the chondrocytes should be alive. Human articular cartilage was obtained from deceased donors and dimethyl sulphoxide (DMSO) was used as the CPA, cooling was at 0.14°C/min and warming at 0.42°C/min. The vehicle solution was CPTes2. A program of increasing DMSO concentration was developed for cooling and this gave satisfactory tissue concentrations but reduction of DMSO concentration during warming was inadequate, resulting in higher tissue concentrations than required. Biomechanical testing indicated a compressive modulus of 9.5±1.3 MPa in LT-processed cartilage, with control values of 11.6±0.8 MPa (p>0.05, Student's t-test). Measurement of GAG synthesis sometimes approached 65% or 85% of control, but the variability of replicate data prevented firm conclusions. Ideally allograft tissue should score 1A or above on the Noyes scale and the donor age should be less than 46 years but the cartilage used in this study did not meet these standards.

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.

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.

Development of a terminally sterilised decellularised dermis.

Many of the decellularised dermis products on the market at present are aspectically produced. NHS Blood and Transplant Tissue Services have developed a method of producing a dCELL human dermis which has been terminally sterilised by gamma irradiation. The terminally sterilised decellularised dermis was compared with cellular tissue and examined for histology, residual DNA content, biomechanical and biochemical properties, in vitro cytotoxicity and in vivo implantation in a mouse model. No alterations in morphology as viewed by light microscopy were observed and DNA removal was 99%. There were no significant changes in ultimate tensile stress or evidence for collagen denaturation or cytotoxicity. The in vivo studies did not indicate any adverse tissue reactions in the mouse model and demonstrated incorporation of dCELL human dermis into the host. Decellularisation, followed by terminal sterilisation with gamma irradiation, is an appropriate method to produce a human dermis allograft material suitable for transplantation.

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.