A Prospective Multicenter Randomized Controlled Trial to Evaluate the Efficacy of Chitosan Hydrogel Paste in Comparison to Commercial Hydroactive Gel as a Wound Bed Preparation.

Background This clinical trial aimed to evaluate the clinical efficacy of chitosan derivative hydrogel paste (CDHP) as a wound bed preparation for wounds with cavities. Methods This study enrolled 287 patients, with 143 patients randomized into the CDHP group (treatment) and 144 patients randomized into the commercial hydroactive gel (CHG) group (control). The granulation tissue, necrotic tissue, patient comfort, clinical signs, symptoms, and patient convenience during the application and removal of the dressing were assessed. Results The study was completed by 111 and 105 patients from the treatment and control groups, respectively. Both groups showed an increasing mean percentage of wound granulation over time when the initial wound size and comorbidity were adjusted (F(10,198) = 4.61; p < 0.001), but no significant difference was found between the groups (F(1,207) = 0.043; p = 0.953). The adjusted mean percentage of necrotic tissue of both groups showed a significant decrease over time (F(10,235) = 5.65; p <0.001), but no significant differences were found between the groups (F (1,244) = 0.487; p = 0.486). Conclusion CDHP is equivalent to CHG and is an alternative in wound management and wound bed preparation for wounds with cavities.

Effects of processing and gamma radiation on mechanical properties and organic composition of frozen, freeze-dried and demineralised human cortical bone allograft.

Bone processing and radiation were reported to influence mechanical properties of cortical bones due in part to structural changes and denaturation of collagen composition. This comparative study was to determine effects of bone processing on mechanical properties and organic composition, and to what extent the radiation damaging after each processing. Human femur cortical bones were processed by freezing, freeze-drying and demineralisation and then gamma irradiated at 5, 15, 20, 25 and 50 kGy. In the compression test, freeze drying significantly decreased the Young's Modulus by 15%, while demineralisation reduced further by 90% (P < 0.05) when compared to the freezing. Only demineralisation significantly reduced ultimate strength of bone by 93% (P < 0.05). In the bending test, both freeze drying and demineralisation significantly reduced the ultimate strength and the work to failure. Radiation at 25 kGy showed no effect on compression for ultimate strength in each processing group. However, high dose of 50 kGy significantly reduced bending ultimate strength by 47% in demineralisation group. Alterations in collagen in bones irradiated at 25 and 50 kGy showed by the highest peak of the amide I collagen in the Fourier Transfer Infra-Red spectra indicating more collagen was exposed after calcium was removed in the demineralised bone, however radiation showed no effect on the collagen crosslink. The study confirmed that demineralisation further reduced the ability to resist deformation in response to an applied force in freeze-dried bones due to calcium reduction and collagen composition. Sterilisation dose of 25 kGy has no effect on mechanical properties and collagen composition of the processed human cortical bone.

Deep-Freezing Temperatures During Irradiation Preserves the Compressive Strength of Human Cortical Bone Allografts: A Cadaver Study.

BACKGROUND: Gamma irradiation, which minimizes the risk of infectious disease transmission when human bone allograft is used, has been found to negatively affect its biomechanical properties. However, in those studies, the deep-freezing temperature during irradiation was not necessarily maintained during transportation and sterilization, which may have affected the findings. Prior reports have also suggested that controlled deep freezing may mitigate the detrimental effects of irradiation on the mechanical properties of bone allograft. QUESTION/PURPOSE: Does a controlled deep-freezing temperature during irradiation help preserve the compressive mechanical properties of human femoral cortical bone allografts? METHODS: Cortical bone cube samples, each measuring 64 mm3, were cut from the mid-diaphyseal midshaft of five fresh-frozen cadaver femurs (four male donors, mean [range] age at procurement 42 years [42 to 43]) and were allocated via block randomization into one of three experimental groups (with equal numbers of samples from each donor allocated into each group). Each experimental group consisted of 20 bone cube samples. Samples irradiated in dry ice were subjected to irradiation doses ranging from 26.7 kGy to 27.1 kGy (mean 26.9 kGy) at a deep-freezing temperature below -40°C (the recommended long-term storage temperature for allografts). Samples irradiated in gel ice underwent irradiation doses ranging from 26.2 kGy and 26.4 kGy (mean 26.3 kGy) in a freezing temperature range between -40°C and 0°C. Acting as controls, samples in a third group were not subjected to gamma irradiation. The mechanical properties (0.2% offset yield stress, ultimate compression stress, toughness, and the Young modulus) of samples from each group were subsequently evaluated via axial compression loading to failure along the long axis of the bone. The investigators were blinded to sample group during compression testing. RESULTS: The mean ultimate compression stress (84 ± 27 MPa versus 119 ± 31 MPa, mean difference 35 [95% CI 9 to 60]; p = 0.005) and toughness (3622 ± 1720 kJ/m3 versus 5854 ± 2900 kJ/m3, mean difference 2232 [95% CI 70 to 4394]; p = 0.009) of samples irradiated at a higher temperature range (-40°C to 0°C) were lower than in those irradiated at deep-freezing temperatures (below -40°C). The mean 0.2% offset yield stress (73 ± 28 MPa versus 109 ± 38 MPa, mean difference 36 [95% CI 11 to 60]; p = 0.002) and ultimate compression stress (84 ± 27 MPa versus 128 ± 40 MPa, mean difference 44 [95% CI 17 to 69]; p < 0.001) of samples irradiated at a higher temperature range (-40°C to 0°C) were lower than the nonirradiated control group samples. The mean 0.2% offset yield stress (73 ± 28 MPa versus 101 ± 28 MPa, mean difference 28 [95% CI 3 to 52]; p = 0.02; effect size = 1.0 [95% CI 0.8 to 1.2]) of samples irradiated at higher temperature range (-40°C to 0°C) were no different with the numbers available to those irradiated at deep-freezing temperature. The mean toughness (3622 ± 1720 kJ/m3 versus 6231 ± 3410 kJ/m3, mean difference 2609 [95% CI 447 to 4771]; p = 0.02; effect size = 1.0 [95% CI 0.8 to 1.2]) of samples irradiated at higher temperature range (-40°C to 0°C) were no different with the numbers available to the non-irradiated control group samples. The mean 0.2% offset yield stress, ultimate compression stress, and toughness of samples irradiated in deep-freezing temperatures (below -40°C) were not different with the numbers available to the non-irradiated control group samples. The Young modulus was not different with the numbers available among the three groups. CONCLUSION: In this study, maintenance of a deep-freezing temperature below -40°C, using dry ice as a cooling agent, consistently mitigated the adverse effects of irradiation on the monotonic-compression mechanical properties of human cortical bone tissue. Preserving the mechanical properties of a cortical allograft, when irradiated in a deep-freezing temperature, may have resulted from attenuation of the deleterious, indirect effects of gamma radiation on its collagen architecture in a frozen state. Immobilization of water molecules in this state prevents radiolysis and the subsequent generation of free radicals. This hypothesis was supported by an apparent loss of the protective effect when a range of higher freezing temperatures was used during irradiation. CLINICAL RELEVANCE: Deep-freezing temperatures below -40°C during gamma irradiation may be a promising approach to better retain the native mechanical properties of cortical bone allografts. A further study of the effect of deep-freezing during gamma radiation sterilization on sterility and other important biomechanical properties of cortical bone (such as, tensile strength, fracture toughness, and fatigue) is needed to confirm these findings.

Does bone marrow aspirate help enhance the integration of gamma irradiated allograft bone?

Bone allografts donated by other individuals offer a viable alternative to autograft. Risks of disease transmission are overcome by sterilizing the bone; unfortunately sterilization methods generally affect bone functional properties including osteogenic potential and biomechanical integrity. This study aimed to determine any enhancement effect when gamma sterilised allografts was impregnated with autologous bone marrow in improving the rate and quality of integration in metaphyseal-tibial defects of rabbits. Almost all subjects showed 50% of the defect being covered by new bones by the third week and smaller residual defect size in the treated group at the fifth week. Hounsfield units at the defect site showed increasing healing in all samples, with the treated group having an apparent advantage although insignificant (p > 0.05). In the histopathological score evaluating healing over cortical and cancellous bone at the fracture site showed only slight variations between the groups (p > 0.05). Therefore no enhanced healing by the autologous bone marrow was observed when added to the bone allografts in treating the unicortical defects.

Improving knowledge of doctors and paramedics through effective bone procurement workshop: A cognitive approach.

BACKGROUND: Procurement of bone allograft must be performed by trained personnel. Improper handling and lack of knowledge during bone procurement will lead to contamination hence jeopardizing quality of the procured bones and expose bone recipients to risks of infection in post-operative phase. Bone procurement workshop is the fundamental training programme to enhance skill among personnel who has been or will be involved in bone procurement. This study evaluated the effectiveness of the workshop contents including teaching materials by assessing the knowledge on bone procurement among the participants before and after the workshop. METHODS: Bone procurement workshop was held for 2 days for doctors and paramedics. The knowledge on bone procurement was evaluated in pre- and post-assessments by answering self administration questionnaire before and after the workshop, respectively. RESULTS: A total of 50 participants comprised of doctors and paramedics attended the workshop however only 15 (55.6%) doctors and 12 (44.4%) paramedics completed the assessments. Overall, the mean total score for the post-assessment (61.4%) was significantly higher (p < 0.05) than that of the pre-assessment score (32.2%). The mean values of correct responses for the post-assessment was significantly higher (p < 0.05) than that of the pre-assessment in all five topics given during the workshop. The correct responses for the pre- and the post- assessments in the respective group of the doctors and paramedics were also statistically significant (p < 0.05). In the pre-assessment, the doctors had the highest score in Surgical Approach & Reconstruction (50%) while the paramedics had the highest score in Donor Screening & Selection Criteria (33.3%). In the post-assessment, the doctors had the highest score in Donor Screening & Selection Criteria (70%) while the paramedics in Packaging & Transportation (65.8%). CONCLUSIONS: The assessment managed to show that the workshop contents and teaching materials were effective in improving the cognitive knowledge of the personnel who would get involved in bone procurement under the National Donation Programme.

Performance of cooling materials and their composites in maintaining freezing temperature during irradiation and transportation of bone allografts.

PURPOSE: Bone allografts supplied by University Malaya Medical Centre Bone Bank are sterilized by gamma radiation at 25 kGy in dry ice (DI) to minimize radiation effects. Use of cheaper and easily available cooling materials, gel ice (GI) and ice pack (IP), was explored. Composites of DI and GI were also studied for the use in routine transportations and radiation process. METHODS: (a) Five dummy bones were packed with DI, GI, or IP in a polystyrene box. The bone temperatures were monitored while the boxes were placed at room temperature over 96 h. Durations for each cooling material maintaining freezing temperatures below -40°C, -20°C, and 0°C were obtained from the bone temperature over time profiles. (b) Composites of DI (20, 15, 10, 5, and 0 kg) and GI were used to pack five dummy bones in a polystyrene box. The durations maintaining varying levels of freezing temperature were compared. RESULTS: DI (20 kg) maintained temperature below -40°C for 76.4 h as compared to 6.3 h in GI (20 bags) and 4.0 h in IP (15 packs). Composites of 15DI (15 kg DI and 9 GI bags) and 10DI (10 kg DI and 17 GI bags) maintained the temperature below -40°C for 61 and 35.5 h, respectively. CONCLUSION: Composites of DI and GI can be used to maintain bones in deep frozen state during irradiation, thus avoiding radiation effects on biomechanical properties. Sterile frozen bone allograft with preserved functional properties is required in clinical applications.

Advances of radiation sterilisation in tissue banking.

Under the auspices of the IAEA tissue banking programme on "Radiation Sterilisation of Tissue Graft" conducted from 1985 to 2004, many scientists and surgeons were involved in various regional research and development (R&D) projects mainly in dealing with radiation dose selection, radiation effects on human tissues and quality system in radiation sterilisation. New findings on radiation effects, tissue processing and preservation were shared during the regional and interregional meetings and workshops. Many tissue banks started to use radiation (25 kGy) to sterilize tissue grafts for tissue safety and efficacy and still continue to use it. The IAEA Code of Practice for Radiation Sterilization of Tissues Allografts developed in 2007 offered simpler methods to conduct radiation dose setting and dose validation experiments for tissue grafts. Advances in dose selection and dose mapping are continued under the quality management system when banks need to be certified to continue their operation. The combination of good tissue processing and preservation as well as good radiation practice will ensure the tissue products are properly sterilised thus safe and of high quality. Experience in meeting challenges in using radiation sterilisation and achievements reported by the tissue bankers are shared here.

Assessing bone banking activities at University of Malaya medical centre.

The main advantage of establishing in-house bone banks is its ability to readily provide allograft bones for local surgeries. Bone procurement activities of our university bone bank during the 10 years of operation were reviewed. Socio-demographic data of donors, types of bone procured, cases of rejected bones and types of allograft bones transplanted are presented. From 179 potential donors, 73 % were accepted with 213 procured bones. Femoral head was the common bone transplanted (45 %), as it was also the most common procured (82 %). Bones were rejected mainly due to non-technical reasons (83 %) rather than positive results of microbiological (13 %) and serological (4 %) tests. Comprehensive data could not be obtained for further analysis due to difficulties in retrieving information. Therefore, quality assurance system was improved to establish more systematic documentations, as the basis of good banking practice with process control hence allowing traceability.

A retrospective study on annual evaluation of radiation processing for frozen bone allografts complying to quality system requirements.

Bone allografts have been used widely to fill up essential void in orthopaedic surgeries. The benefit of using allografts to replace and reconstruct musculoskeletal injuries, fractures or disease has obtained overwhelming acceptance from orthopaedic surgeons worldwide. However, bacterial infection and disease transmission through bone allograft transplantation have always been a significant issue. Sterilization by radiation is an effective method to eliminate unwanted microorganisms thus assist in preventing life threatening allograft associated infections. Femoral heads procured from living donors and long bones (femur and tibia) procured from cadaveric donors were sterilized at 25 kGy in compliance with international standard ISO 11137. According to quality requirements, all records of bone banking were evaluated annually. This retrospective study was carried out on annual evaluation of radiation records from 1998 until 2012. The minimum doses absorbed by the bones were ranging from 25.3 to 38.2 kGy while the absorbed maximum doses were from 25.4 to 42.3 kGy. All the bones supplied by our UMMC Bone Bank were sterile at the required minimum dose of 25 kGy. Our analysis on dose variation showed that the dose uniformity ratios in 37 irradiated boxes of 31 radiation batches were in the range of 1.003-1.251, which indicated the doses were well distributed.

Effects of different doses of gamma irradiation on oxygen and water vapour transmission rate of preserved human amniotic membrane.

Preserved human amniotic membrane either air dried or glycerol preserved has been used effectively to treat superficial and partial thickness wounds without leaving any obvious hypertrophic scar. The preserved amnion, sterilised by ionising radiation, is known as an effective barrier for heat, fluid and protein loss while adheres nicely on wound. Air drying slightly reduced the oxygen transmission rate (OTR) of the amnion and the value significantly dropped after 15 kGy (p < 0.05). Glycerol preservation significantly reduced (p < 0.05) the OTR indicating less oxygen transmitted through the well structured cells of the amnion. Increase in the OTR with the increasing radiation doses up to 35 kGy possibly due to direct effects of radiation that resulted in large intercellular gaps. Both preservation methods significantly increased (p < 0.05) the water vapour transmission rate (WVTR). However, the low WVTR in the air dried amnion at 15 and 25 kGy was postulated due to cross-linking of collagen. Changes in the biophysical properties can be linked to direct and indirect effects of radiation on collagen bundles. The radiation dose of 25 kGy caused no adverse effect on biophysical properties hence it is still acceptable to sterilize both the air dried and the glycerol preserved amnions.

Scanning electron microscopic assessment on surface morphology of preserved human amniotic membrane after gamma sterilisation.

Human amniotic membrane that has been processed and sterilised by gamma irradiation is widely used as a biological dressing in surgical applications. The morphological structure of human amniotic membrane was studied under scanning electron microscopy (SEM) to assess effects of gamma radiation on human amniotic membrane following different preservation methods. The amniotic membrane was preserved by either air drying or submerged in glycerol before gamma irradiated at 15, 25 and 35 kGy. Fresh human amniotic membrane, neither preserved nor irradiated was used as the control. The surface morphology of glycerol preserved amnion was found comparable to the fresh amniotic membrane. The cells of the glycerol preserved was beautifully arranged, homogonous in size and tended to round up. The cell structure in the air dried preserved amnion seemed to be flattened and dehydrated. The effects of dehydration on intercellular channels and the microvilli on the cell surface were clearly seen at higher magnifications (10,000×). SEM revealed that the changes of the cell morphology of the glycerol preserved amnion were visible at 35 kGy while the air dried already changed at 25 kGy. Glycerol preservation method is recommended for human amniotic membrane as the cell morphological structure is maintained and radiation doses lower than 25 kGy for sterilization did not affect the appearance of the preserved amnion.

Gelam (Melaleuca spp.) Honey-Based Hydrogel as Burn Wound Dressing.

A novel cross-linked honey hydrogel dressing was developed by incorporating Malaysian honey into hydrogel dressing formulation, cross-linked and sterilized using electron beam irradiation (25 kGy). In this study, the physical properties of the prepared honey hydrogel and its wound healing efficacy on deep partial thickness burn wounds in rats were assessed. Skin samples were taken at 7, 14, 21, and 28 days after burn for histopathological and molecular evaluations. Application of honey hydrogel dressings significantly enhanced (P < 0.05) wound closure and accelerated the rate of re-epithelialization as compared to control hydrogel and OpSite film dressing. A significant decrease in inflammatory response was observed in honey hydrogel treated wounds as early as 7 days after burn (P < 0.05). Semiquantitative analysis using RT-PCR revealed that treatment with honey hydrogel significantly (P < 0.05) suppressed the expression of proinflammatory cytokines (IL-1α, IL-1ß, and IL-6). The present study substantiates the potential efficacy of honey hydrogel dressings in accelerating burn wound healing.

Sea cucumber (Stichopus hermanii) based hydrogel to treat burn wounds in rats.

Malaysian sea cucumber was incorporated into hydrogel formulation by using electron beam irradiation technique and was introduced as novel cross-linked Gamat Hydrogel dressing. This study investigated whether Gamat Hydrogel enhanced repair of deep partial skin thickness burn wound in rats and its possible mechanism. Wounds were treated with either Gamat Hydrogel, control hydrogel, OpSite® film dressing or left untreated. Skin samples were taken at 7, 14, 21, and 28 days post burn for histological and molecular evaluations. Gamat Hydrogel markedly enhanced wound contraction and improved histological reorganization of the regenerating tissue. Furthermore, the dressing modulated the inflammatory responses, stimulated the activation and proliferation of fibroblasts, and enhanced rapid production of collagen fiber network with a consequently shorter healing time. The level of proinflammatory cytokines; IL-1α, IL-1ß, and IL-6, were significantly reduced in Gamat Hydrogel treated wounds compared with other groups as assessed by reverse transcription-polymerase chain reaction (RT-PCR). In summary, our results showed that Gamat Hydrogel promoted burn wound repair via a complex mechanism involving stimulation of tissue regeneration and regulation of pro-inflammatory cytokines. The resultant wound healing effects were attributed to the synergistic effect of the hydrogel matrix and incorporated sea cucumber.

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Malaysia.

The IAEA was instrumental in developing the first Malaysian tissue bank at University Hospital of Universiti Sains Malaysia (HUSM), Kubang Kerian, Kelantan in early 1990s and it was officiated as National Tissue Bank in 1994. Up to date, 38 government and private hospitals have received a supply from the bank. Bone allografts in term of bone chips, morsalised bone and long bones are procured from Malaysian donors. Almost thirty students from Malaysia graduated in the training courses carried out in Singapore since 1998 at regional and interregional levels. Organ donation is more readily accepted by the public at the moments, perhaps due to the vast promotion and advertisement given by the local newspapers and other media, but gradually tissue donation is catching up as well.