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The decellularization of bovine bone has emerged as a strategy for the repair, replacement, and regeneration of bone defects. To evaluate the effects of a new protocol of bone decellularization and its impact on the structure and collagen scaffold. Cancellous bone from bovine femur was dissected in fragments and decellularized based on protocol of multiple steps. The residual protein levels, histological, morphometric, and scanning electronic microscopy analyses were carried out to evaluate the effects of decellularization and the impact on the structure and collagen scaffold. A cytotoxicity assay was performed. Residual protein analysis showed an important removal of bone marrow components and cell debris from the bone. Sections revealed that collagen fibers presented integrity and absence of cells in the decellularized bone. Sirius Red-stained sections of collagen fiber collagen matrix were maintained after decellularization. Scanning electron microscopy revealed that the main bone structure, despite being irregular, was maintained in both groups, with no significant visual differences between the surface characteristics according to the groups. Decellularized bovine bone demonstrated a degree of toxicity of 3, indicating moderate reactivity. The present data demonstrate that the main bone structure was maintained. Additionally, the chemical and physical treatments were able to remove cellular debris, and extracellular matrix architecture and collagen were preserved. However, the tissue showed moderate toxicity., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Musculoskeletal allografts are used in reconstructive procedures, however, the risk of contamination with potential pathogens is possible, and safe transplantation requires multiple processing considerations. Hydrogen peroxide (H 2 O 2 ) has commonly been used in bone washing because it can remove donor cells and eliminate antigens, pathogens, or cytotoxic agents from the matrix. The aim of this study was to evaluate the quantitative activity of H 2 O 2 in a model of bone contamination with a high bacterial load to define the bioburden reduction. Twelve bone disc models were artificially contaminated with Staphylococcus aureus. The bones were treated with a washing process composed by antibiotics, 30% hydrogen peroxide, and 70% alcohol. Tryptic Soy Agar plates were directly inoculated with 100µL of each step of the washing process and colonies were counted in CFU/mL. Scanning electron microscopy was used for bone structural analysis before and after the washing process. After antibiotics, there was a drop of less than 1 log for cancellous bone and almost 1 log for cortical bone. However, after H 2 O 2 , there as a drop of 3 logs for cortical (p = 0.007), and 2 logs for cancellous bone (p = 0.063). The use of alcohol did not change the bioburden following H 2 O 2 in cancellous and cortical bone. Despite the important drop of bacterial load, H 2 O 2 was not enough to completely eradicate bacterial with this model of bioburden. H 2 O 2 is useful in decontamination, but antibiotics have little activity, and alcohol is useless. The process is useful in decontamination up to 3 logs of bioburden., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Residual chemicals that are presented during tissue processing in human tissue banks can be a risk for the allograft recipient. Determine the residual concentrations of the antibiotics and detergent used in the process of human decellularized tissue-engineered heart valves stored in isotonic saline solution up to 18 months. A total of 24 human decellularized allografts were stored in sterile sodium chloride and analyzed immediately after the decellularization process (0 months) and after storage for 6, 12, and 18 months, which includes the use of sodium dodecyl sulfate (SDS) and antibiotics (cefoxitin, vancomycin hydrochloride, lincomycin hydrochloride, polymyxin B sulfate). These valves were used for suitability tests, the zone of inhibition evaluation, and direct contact cytotoxicity assay. The stock solution from 32 valves was used for LC-MS/MS analysis of antibiotics and SDS. Tissue samples from decellularized valves showed a zone of inhibition formation for S. aureus and B. subtilis, suggesting the presence of an inhibitory molecule in the tissue. Cytotoxicity tests were negative. Polymyxin B, vancomycin, and SDS were detected and quantified in human decellularized aortic and pulmonary allografts during all periods of the study. There were no traces of residual cefoxitin and lincomycin in the tissue stock solution. We found residual concentrations of the antibiotics and detergent used in the process of human decellularized tissue-engineered heart valves stored in isotonic saline solution up to 18 months.

The contamination of the transport solution used in cardiovascular allografts can occur from different sources. Risk factors associated with positive microbiological test of transport solution have not been reported previously. This study aimed to determine the risk factor for contamination of transport solution used in the heart valve allografts stored in a Brazilian tissue bank. This retrospective study was conducted on all donors of cardiovascular allografts stored in a tissue bank from December 2008 to December 2017. Microbiological cultures for aerobic and anaerobic bacteria, fungi/yeasts were carried out in TS. Clinical variables were included. From 1001 transport solution, 52% were contaminated. A total of 770 microorganisms were identified, and Staphylococcus spp. was identified in 248 isolates (32.2%). Skin bacteria from skin microbiota were the most commonly identified microorganisms (Staphylococcus spp., Cutibacterium spp., Corynebacterium spp., and Bacillus spp.), occurring in 49.6%. The presence of a diagnosis of healthcare-associated infection was not associated with skin contamination (odds ratio [OR] 0.62 [0.41-0.94]; p = 0.014). Conditions like fever, use of antibiotics, and leukocytosis were less likely associated with contamination of transport solution. A longer warm ischemic time was associated with higher frequency of contamination. In the multivariable analysis, warm ischemic time was independently associated with contamination, and antibiotic therapy was a factor that decreased the rate of contamination (p < 0.05). Contamination of transport solution is associated with modifiable risk factors, such as warm ischemic time. Measures to minimize contamination should be employed to avoid unnecessary tissue discharges.

Most tissue banks use the conventional method; however, the automated method has advantages over the conventional method. The aim of this study was to compare the conventional and automated methods of culture in human cardiac tissue using an artificial contamination model. Myocardial samples were contaminated with sequential concentration (10 4 to 10 -1  CFU/mL) with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Cultures were obtained from solution were the fragment was immersed and minced tissue, before and after the routine decellularization solution, with automated and conventional culture methods. Automated and conventional methods were compared and a p value ≤ 0.05 was considered significant. Staphylococcus aureus presented a significantly higher growth in the automated method, as well as faster than the conventional (p < 0.05). The positivity for growth in the automated method was higher in concentrated inoculum (> 10 2  CFU/mL) (p < 0.05). The growth in the automated method was significantly faster than conventional when inoculum concentration was above 10 3  CFU/mL. The automated culture method is faster than conventional method with a higher positivity in a contaminated model of myocardial and transport solution used in tissue banks.

All cardiac allograft tissues are under potential contamination, requiring a validated terminal sterilization process or a minimal bioburden. The bioburden calculation is important to determine the bacterial burden and further decontamination and disinfection strategies for the valve processing. The aim of this study was to determine the bioburden from transport solution (TS) of heart valves obtained from non-heart-beating and heart-beating donors in different culture methods. The bioburden from TS was determined in 20 hearts donated for valve allograft tissue using membrane filter (MF) and direct inoculation. Tryptic soy agar and Sabouraud plates were incubated and colonies were counted. Ninety-five percent of samples from this study were obtained from heart-beating donors. The warm ischemic time period for heart was 1.06 ± 0.74 h and the cold ischemic time period was 25.66 ± 11.16 h. The mean TS volume was 232.68 ± 96.67 mL (48.5-550 mL). From 20 samples directly inoculated on TSA agar plates, 2 (10%) were positive. However, when MF was used, from 20 samples in TSA, 13 (65%) were positive with a mean count of 1.36 ± 4.04 CFU/mL. In Sabouraud plates, the direct inoculation was positive in 5 samples (25%) with a mean count of 0.24 ± 0.56 CFU/mL. The use of MF increased the positivity to 50% (10 samples from a total of 20) with a mean of 0.28 ± 0.68 CFU/mL. The positivity was superior using MF in comparison with direct inoculation (p < 0.05). The bioburden of TS is low and MF is the technique of choice due to higher positivity.