Effect of glycerol concentrations and temperatures on epidermal growth factor protein expression in preserved canine amniotic membrane Abstract Amniotic membrane has been widely applied as a biological graft in both medical and veterinary practice. In ophthalmology, epidermal growth factor (EGF) in human amniotic membrane (HAM) promotes corneal epithelial cell proliferation and migration, thus it facilitates corneal wound healing. In dogs, with limited cryopreserved HAM availability, different cold glycerol preserving protocols have been developed for the storage canine amniotic membrane (CAM). This study aimed to study protein expression of EGF in CAM preserved with different concentrations of glycerol and storage temperatures, using enzyme-linked immunosorbent assay. CAM preserved in 50% glycerol and 99.5% glycerol and kept at 4 and − 20 °C for 7–30 days were compared. We found that preserving membrane with 50% glycerol at − 20 °C has significantly higher EGF protein expression compared with that at 4 °C (p < 0.05). There was a trend that the storage in 50% glycerol achieved higher EGF protein expression than 99.5% glycerol at both 4 °C and − 20 °C. In conclusion, 50% glycerol at − 20 °C was the best condition to preserve CAM in our study. Therefore, there is likely an alternative method to maintain level of EGF protein expression in preserved CAM.

Professional burnout and its correlates in Polish donor transplant coordinators Abstract Job demands-resources (JD-R) model of professional burnout states that job demands predict the feeling of exhaustion, and lack of job resources—disengagement from work. This research project investigated professional burnout and it correlates, including sex, death anxiety, and relationship status in 108 Polish donor transplant coordinators involved in organ, tissue, and cell transplantations. This study employed the Polish version of the Oldenburg Burnout Inventory which follows the JD-R model, the Psychosocial Working Conditions Questionnaire—a Polish instrument based on the model of job stress proposed by Karasek—and the Polish version of the Fear of Death and Dying Questionnaire. The results were suggestive of average levels of job stress and burnout in the studied population, with men being more disengaged than women. Participants who were in relationship had significantly higher levels of exhaustion than those who were single. Exhaustion was positively correlated with years of working as a transplant coordinator but not with participants’ age. Multiple negative correlations were detected between exhaustion/disengagement and different aspects of job control, social support, and well-being. Moreover, positive correlations between different components of fear of death and dying and exhaustion were detected. Our findings, linking fear of death and dying with some aspects of professional burnout in transplant coordinators, suggest that a pre-employment screening for the level of death anxiety in candidates for transplant coordinators could be useful as this job provides chronic exposure to mortality cues.

Establishing a correlation between RIN and A 260/280 along with the multivariate evaluation of factors affecting the quality of RNA in cryopreserved cancer bio-specimen Abstract Biomarkers are biological characteristic to measure and evaluate normal and pathological states. To define expression-based molecular biomarkers, high-quality tissue samples are a prerequisite for the preparation of standard RNA. It is already known that RIN number defines the RNA quality, however its relation with A260/280 ratio and Ct value is not defined clearly; therefore, understanding an association will provide a reliable method for describing RNA quality. Multiple cryopreserved human tumor tissue types from ACTREC Biorepository and TMH-INTTR were analyzed for the effect of storage time on RNA quality. The RNA from tumor samples were isolated and analyzed by RIN, A260/280 ratio, and Ct value to establish inter-relationships. Around 50% samples had a RIN of ≥ 6.9 and A260/280 ≤ 2.04; 27% had a RIN ≥ 5 and A260/280 ≤ 2.08, and remaining 23% displayed RIN < 5 and A260/280 > 2.08. However, the RNA quality has no association with the storage period of tissue samples. Moreover, all samples which had A260/280 ≤ 2.08 showed acceptable Ct values of 17–24. The data clearly suggests that the A260/280 ratio is able to predict RNA quality. To the best of our knowledge, this is the first Indian report analyzing the labile nucleic acid-RNA quality from different cancer tissue types cryopreserved for diverse time periods. In conclusion, RIN and A260/280 ratio can help in predicting the quality of RNA independently; however, both together provide better assurance for further downstream processing. Graphic abstract

Verifying measurements of residual calcium content in demineralised cortical bone Abstract Calcium contents of demineralised human cortical bone determined by titrimetric assay and atomic absorption spectrophotometry technique were verified by comparing to neutron activation analysis which has high recovery of more than 90%. Conversion factors determined from the comparison is necessary to correct the calcium content for each technique. Femurs from cadaveric donors were cut into cortical rings and demineralised in 0.5 M hydrochloric acid for varying immersion times. Initial calcium content in the cortical bone measured by titration was 4.57%, only 21% of the measurement by neutron activation analysis; while measured by atomic absorption spectrophotometer was 13.4%, only 61% of neutron activation analysis. By comparing more readings with the measurements by neutron activation analysis with 93% recovery, a conversion factor of 4.83 was verified and applied for the readings by titration and 1.45 for atomic absorption spectrophotometer in calculating the correct calcium contents. The residual calcium content started to reduce after the cortical bone was demineralised in hydrochloric acid for 8 h and reduced to 13% after 24 h. Using the linear relationship, the residual calcium content could be reduced to less than 8% after immersion in hydrochloric acid for 40 h. Atomic absorption spectrophotometry technique is the method of choice for calcium content determination as it is more reliable compared to titrimetric assay.

Decellularization, cross-linking and heparin immobilization of porcine carotid arteries for tissue engineering vascular grafts Abstract Tissue engineering vascular grafts (TEVGs) have the potential to replace small-diameter grafts in bypass surgery which is good news for patients with cardiovascular disease. Decellularized arteries can be ideal TEVGs because their natural three-dimensional structures support the migration of host cells and vascular remodeling. There are many methods for decellularization without a standard protocol. In this study, a combination of Triton X-100 and sodium dodecyl sulfate (SDS) were used to prepare decellularized arteries. However, decellularization may damage the biochemical and mechanical properties to some degree. We used the cross-linking agents N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to improve mechanical properties and immobilize heparin to inhibit thrombogenesis. Histological analysis, scanning electron microscopy, biomechanical properties test, determination of immobilized heparin, active partial thrombin time assay, and subcutaneous embedding experiment were used to evaluate the efficiency of decellularization and the efficacy of heparinized cross-linked vascular scaffold. Results showed 1% Triton X-100 combined with 0.3% SDS can decellularize successfully. EDC and NHS cross-linking can improve the mechanical properties, reduce the inflammatory reaction and slow the degradation time. Heparin immobilized on the scaffolds can inhibit thrombogenesis effectively. This study indicated the heparinized cross-linked vascular scaffolds may be ideal scaffolds for TEVGs.

Attitudes of Asian Parkinson patients towards brain donation Abstract Histopathological examination of brain tissue is required for better understanding of neurodegenerative conditions such as Parkinson’s disease and related disorders. However, patient willingness remains the greatest hurdle hampering participation in brain donation for research. While there is extensive research being conducted on the subject in West, to the best of our knowledge, there are no studies done in this regard in Asia. This cross-sectional survey was conducted on 105 Parkinson’s disease patients to assess their knowledge, beliefs and attitude towards brain donation in an Asian population. The majority of the participants (78%) acknowledged the importance of donation of brain for research, and 70% believed that their donated brain samples would be handled professionally. Fifty percent participants were willing to consider donating their brain for research. Motivating factors for brain donation included altruism (87%) and contribution to advance medical knowledge (80%). Common reasons for unwillingness towards brain donation were stress for family (30%), disfigurement of body (25%), and having a conservative mindset (23%). About one-third of the participants preferred to be approached for brain donation after their first clinic visit. Most patients preferred either their treating neurologists (66%) or research staff (18%) to discuss brain donation with. Participation for brain donation may be increased further with greater patient and public education to overcome misconceptions and change mindsets.

Objective evaluation of healing and esthetic outcome of root coverage procedure using chorion membrane: a case series Abstract The ultimate goal of any periodontal plastic surgery aimed to treat gingival recession is predictable recession coverage and esthetic outcome. Due to the post-operative morbidity and discomfort related to subepithelial connective tissue graft, various other methods have been introduced including the use of fetal membranes. The fetal membranes have shown promising results in medicine and recently in the field of regenerative dentistry and could possibly become a viable alternative to autogenous grafts. This article reports a series of cases of Miller’s class I gingival recession that were treated by chorion membrane combined with coronally advanced flap and evaluated using objective variables for post-operative healing and esthetics. Nine systemically healthy subjects with ten Miller’s class I buccal gingival recession were included in the study. Clinical parameters were recorded at baseline, 3 months and 6 months post-surgery; wound healing index (WHI) was recorded one week post-surgery. At the end of 6 months, the percentage of root coverage and the root coverage esthetic score (RES) were calculated. The results showed statistically significant (p < 0.05) improvement in most of the parameters. The mean percentage of root coverage obtained was 62.20 ± 21.99% ranging from 33.3 to 100%. The WHI showed excellent healing score and RES assessed at the end of six months showed superior esthetic results. The chorion membrane along with coronally advanced flap is a predictable procedure for root coverage with better post surgical healing and superior esthetics.

The effect of different cross-linking conditions of EDC/NHS on type II collagen scaffolds: an in vitro evaluation Abstract The purpose of this paper is to analyze the properties of porcine cartilage type II collagen scaffolds crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy-succinamide (EDC/NHS) under different conditions. The porous EDC/NHS-crosslinked scaffolds were obtained through a two-step freeze-drying process. To determine the optimal crosslinking condition, we used different solvents and various crosslinking temperatures to prepare the scaffolds. Three crosslinking solutions were prepared with different solvents, photographs were taken with a flash in the darkroom, and light transmission was observed. Type II collagen was crosslinked on a horizontal shaker at a speed of 60 r/min according to the above grouping conditions, and then the structural change of the scaffold in each group was observed. To investigate the swelling ratio and the in vitro degradation of the collagen scaffold, tests were also carried out by immersion of the scaffolds in a PBS solution and digestion in type II collagenase, respectively. The influence of the scaffolds on the proliferation of chondrocytes was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The morphology of the crosslinked scaffolds cocultured with chondrocytes was characterized by a scanning electron microscope. The results proved that 75% alcohol and a crosslinking temperature of 37 °C are recommended. Collagen fibrils are more densely packed after crosslinking with EDC/NHS and have a more uniform structure than that of noncrosslinked ones. The EDC-crosslinked scaffolds possessed excellent mechanical property and biocompatibility.

Why people donate their brain to science: a systematic review Abstract The acquisition of brain tissue for research purposes is an important endeavour in research on ageing, pathological diagnosis, and the advancement of treatment of neurological or neurodegenerative diseases. While some tissue samples can be obtained from a living patient, the procurement of a whole brain requires the donation from people after their death. In order to promote positive attitudes towards brain donation, it is essential to understand why people do or do not donate their brain to medical research. In 2018 we undertook a systematic review of the international literature concerning people’s attitudes, motivations, and feelings about brain donation. Five electronic databases were searched: Scopus, PsycINFO, Embase, Medline, and Google Scholar. Search terms included: (“brain donor*” OR “brain donation” OR “brain banking” OR “banking on brain”) AND (attitude* OR motivation* OR decision*”) AND (LIMIT-TO “human”) AND (LIMIT-TO (LANGUAGE, “English”)). Articles were analysed using the Framework for Assessing Qualitative Evaluations and a meta-ethnographic approach. Fourteen articles were included for review. The findings suggest four universal factors informing a person’s decision to donate their brain: (1) contextual knowledge, (2) conceptual understandings, (3) family/friends matter, and (4) personal experience, time and process. The findings also indicate that the way healthcare professionals present themselves can influence people’s feelings and attitudes towards brain donation. Healthcare and research professionals who are involved in brain donation processes must be mindful of the complex and multiple factors that influence donation outcomes. Effective and sensitive communication with potential donors and their family/friends is paramount.

Rolling–sliding load decreases the loss of chondrocyte viability and the mechanical properties of cartilage explants preserved in vitro Abstract The viability of cartilage explants preserved in vitro decreases with time, which limits its use for transplantation. The effect of mechanical stimulation to cartilage explants in vitro is unknown. In this study, we observed the effects of mechanical stimulation on chondrocyte viability and the mechanical properties of cartilage explants preserved in vitro using a rolling–sliding loading device designed by us, and the optimal stimulation protocol was established. A cylindrical osteochondral mass drilled on the femoral condyle of a healthy pig was divided into two groups (loading group and control group), and changes in the chondrocyte survival rate, matrix composition and cartilage biomechanical properties was observed at different time points. Additionally, the mRNA expression of the apoptosis-related proteins caspase-3/Bax/Bcl-2, the cytoskeletal proteins actin/vimentin, and the matrix-related protein MMP13 were detected. The loading group exhibited delayed collagen and aggrecan degeneration and improved chondrocyte viability for three days. Protein and mRNA detection showed that apoptotic factors such as caspase-3 and Bax decreased rapidly in cartilage tissue after loading. The cytoskeletal proteins actin and vimentin showed no significant changes in mRNA expression in the control group, but was significantly higher in the loading group. MMP-13 mRNA expression was significantly higher in both the control group and loading group. Overall, this study suggests that suitable mechanical stimulation decreases the loss of chondrocyte viability and the mechanical properties of cartilage explants in vitro and improves cartilage preservation.