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Objective: This study aimed to assess the complication rate during and 24 hours after cryoablation in lung, bone, and soft tissue tumors., Methods: We reviewed complications in a total of 85 consecutive patients who underwent cryoablation using a liquid nitrogen-based system in various lesions between April 2017 and October 2022. There were no liver and renal lesions. Complications were categorized using the Society of Interventional Radiology classification., Results: Eighty-five patients were treated for 96 lesions in the bone (36.4%; 35 of 96), lung (18.8%; 18 of 96), and soft tissue (44.8%; 43 of 96). The primary technical success rate was 97.7% (83 of 85). The total grade 2 and 1 complication rates were 5.2% (5/96) and 20.8% (20/96), respectively. Two patients had asymptomatic pulmonary embolisms incidentally noted at the 24-hour follow-up computed tomography (grade 2). The most frequent complications were simple and hemorrhagic pleural effusions (18.7%; 18 of 96). Lung procedures had the highest complication rate, where 13 patients (72.2%; 13 of 18) reported complications, including 2 cases of symptomatic hydropneumothorax requiring drainage (grade 2) and an additional 2 days of hospital stay. Eight patients (24.2%; 8 of 33) with bone lesions and 4 (9.3%; 4 of 43) with soft tissue lesions experienced complications., Conclusion: Cryoablation using a liquid nitrogen-based system is safe, with only minor complications observed., Advances in Knowledge: This study provides data on the safety of liquid nitrogen-based percutaneous cryoablation in tumors located in lung, in bones and in soft tissues. Despite using larger diameter cryoprobes than those typically reported with argon-based system, our experience shows that complications are mostly low and comparable in frequency and severity., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Institute of Radiology.)

Background: Actinic keratoses (AK) may progress into squamous cell carcinoma. Many combination therapies were used to improve the clearance rate. However, there are limited studies on the efficacy and safety of lesion-directed Liquid nitrogen cryotherapy combined with field-directed photodynamic therapy (PDT)., Objective: This study is aimed to determine the efficacy and safety of Liquid nitrogen cryotherapy combined with PDT in Chinese patients with actinic keratosis., Methods: A total of 61 patients with AK were selected at the Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College between August 2018 and August 2023. They were randomly divided into PDT plus Liquid nitrogen cryotherapy group (LN-PDT) and control PDT group (C-PDT) with 30 patients and 31 patients, respectively. 27 patients in the LN-PDT group and 28 patients in the C-PDT group completed the clinical sessions. Efficacy, adverse effects, and cosmetic outcomes were recorded and compared between the two groups., Results: 3 months after the final treatment, the clearance rates for total AK lesions were 97.4 % (449/461) in the LN-PDT group and 93.4 % (456/488) in the C-PDT group (P < 0.05). For grade I AK lesions, the clearance rates were 99.2 % (234/237) for the LN-PDT group and 98.3 % (237/241) for the C-PDT group (P >0.05). For grade II lesions, the clearance rates were 97.5 % (156/160) for the LN-PDT group and 91.9 % (172/186) for the CPDT group (P < 0.05). For grade III lesions, the clearance rates of the LN-PDT and C-PDT groups were 87.5 % (58/64) and 78.7 % (47/61), respectively (P < 0.05). The two groups had no significant differences in pain, erythema, edema, hyperpigmentation and scarring. Cosmetic outcomes mainly were excellent or good in both groups with no significant difference., Conclusion: Liquid nitrogen cryotherapy combined with PDT showed higher efficacy on grade II and grade III AK lesions than PDT alone. The two groups have similar adverse effects and cosmetic outcomes., (Copyright © 2024. Published by Elsevier B.V.)

Fish swim bladder (FSB) is a type of traditional nutraceutical, but the lack of high-quality drying methods limits its premium market development. In order to obtain optimal-quality dried FSBs from Chinese longsnout catfish, the effects of liquid nitrogen pre-freezing (LNF) and drying on the physical properties and flavor of FSB were evaluated. Four methods were used for FSB drying, including natural air-drying (ND), hot-air-drying (HD), LNF combined with freeze-drying (LN-FD), and LNF combined with HD (LN-HD). Color, collagen content, rehydration ratio, textural properties, and flavor characteristics (by GC-IMS, E-nose, and E-tongue) were measured to clarify the differences among four dried FSBs. The results showed that ND cannot effectively remove moisture from FSB as the final product showed a stronger sourness in taste. HD led to a decrease in the collagen content and the collapse of the fiber structure in FSB. Compared to HD, LN-HD showed a higher collagen content (0.56 g/g) and a different flavor fingerprint. FSB treated by LN-FD had better physical qualities in terms of an attractive color, a high collagen content (0.79 g/g), low shrinkage, a higher rehydration ratio (2.85), and a soft texture, while also possessing richer characteristic flavors. The application of LN-FD may help the optimization of the nutrition level, rehydration ability, mouthfeel, and flavor of dried FSB.

Subcooled liquid nitrogen and nitrogen slush are often considered for high-speed cooling, but their preparation and maintenance are not easy. To address this issue, a unique device was designed to prepare subcooled liquid argon (SLA) using liquid nitrogen (LN). The cooling process was mathematically modeled to predict the preparation time. If the interlayer space between LN and liquid argon is filled with nitrogen gas, liquid argon could be cooled to 3.5 K subcooling within 1 h. If the interlayer is filled with air, 2 h are required to achieve the same subcooled state. An additional 1000 mL of LN was required for the preparation of 600 mL of 3.5 K SLA. The cooling tests of 3 μL microdroplets in 3 mm-6 mm capillary quartz tubes were duplicated to evaluate the potential of SLA. It was found that the cooling rate of microdroplet in the 3.5 K subcooled SLA is very close to that in the 3 K subcooled LN, higher than that in the saturated LN. The convenience of preparation and maintenance of SLA can make it good choice of cryogen for cryopreservation of biomaterials., Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest exists., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

This research has dealt with the simulation of liquid nitrogen cavitation inside a convergent nozzle. This is important in cryogenic industrial applications. So in this study, computational fluid dynamics methods have been used for simulating the cavitation phenomenon. The Two-phase model in this research has been a hybrid/mixed model. Also, k- ε turbulence model has been employed in realizable state. For meshing the nozzle geometry, Gambit software has been used, while for numerical simulation, Ansys Fluent software has been employed. For simulation of cavitation, Schnerr and Sauer cavitation model has been utilized. This research has also examined the effect of changing the nozzle outlet diameter and the impact of changing the pressure difference in the inlet and outlet of the nozzle on the cavitation. As a novelty and unlike what would have been expected based on the Bernoulli effect, the results obtained from the simulation showed that the increase/decrease in the nozzle's outlet diameter resulted in an enhanced/diminished extent of cavitation in the nozzle's outlet region. Also, the increase/decrease of the pressure difference in the input and output of the nozzle would lead to a higher/lower extent of cavitation. This research also found that the effect of altering the nozzle's outlet diameter on the extent of cavitation has been far higher than the effect of changing pressure difference in its inlet and outlet. The results also indicated that upon reduction of the nozzle's outlet diameter from the base state (1.02 mm) by 10, 20, 30, 40, and 50 %, the volume fraction of the vapor diminished by 22.23, 43.029, 60.66, 74.73, and 87.16 % respectively. Finally, with the increase in the nozzle's outlet diameter from the base state (1.02 mm) by 10, 20, 30, 40, and 50 %, the volume fraction of the vapor increased by 26.83, 55.27, 84.47, 117.12, and 149.31 % respectively., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The gold standard treatment for primary bone sarcomas has been surgical resection with wide margins. However, there is no consensus regarding an optimal method for limb salvage reconstruction. In 2005, a technique for recycling resected bone after intraoperative treatment with liquid nitrogen was described. This technique has been reported to have a spectrum of advantages; nonetheless, acceptance for routine use has been limited, primarily for fear of local recurrence. A systematic search of the literature using PubMed and Google Scholar was performed. Full-text articles published between 2008 and 2023 were included if the study presented sufficient information regarding patients with a diagnosis of a primary bone sarcoma of the limbs or pelvis who had undergone reconstruction with liquid nitrogen recycled autografts. Sixteen studies that included 286 patients met criteria for analyses. Local recurrence occurred in 25 patients (8.7 %) during the first 4 years following limb salvage reconstruction using recycled autografts for treatment of primary bone sarcomas, which compares favorably to the 15-30 % local recurrence rates reported for patients undergoing limb salvage reconstruction using artificial implants. Systematic synthesis of the current evidence regarding local recurrence rates following use of the liquid nitrogen recycled autograft technique for limb salvage reconstruction after bone sarcoma resection suggests a favorable comparison to other limb salvage reconstruction options. As such, this technique warrants further consideration as a viable option for indicated patients based on relative advantages regarding costs, availability, and biologic and surgical reconstruction benefits., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This is to certify that all authors have seen and approved the final version of the manuscript being submitted. The article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere., (© 2024 The Author(s).)

Due to its boiling point of -196 °C, liquid nitrogen is a cryogenic substance which is commonly used in many industries for its cooling properties. However, this extreme cooling capability means that it also has the potential to inflict severe full-thickness burns. Despite its widespread use in the workplace, very little has been described in the literature regarding complex reconstruction of liquid nitrogen burns. We present a case that is unique, not only in its unusual mechanism of injury but also in that it is the first described case of free tissue reconstruction of cryogenic burns., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Burn Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Progressive loss of plant diversity requires the protection of wild and agri-/horticultural species. For species whose seeds are extremely short-lived, or rarely or never produce seeds, or whose genetic makeup must be preserved, cryopreservation offers the only possibility for long-term conservation. At temperatures below freezing, most vegetative plant tissues suffer severe damage from ice crystal formation and require protection. In this review, we describe how increasing the concentration of cellular solutes by air drying or adding cryoprotectants, together with rapid cooling, results in a vitrified, highly viscous state in which cells can remain viable and be stored. On this basis, a range of dormant bud-freezing, slow-cooling, and (droplet-)vitrification protocols have been developed, but few are used to cryobank important agricultural/horticultural/timber and threatened species. To improve cryopreservation efficiency, the effects of cryoprotectants and molecular processes need to be understood and the costs for cryobanking reduced. However, overall, the long-term costs of cryopreservation are low, while the benefits are huge.

This study examines a rare case of frostbite on the hands caused by liquid nitrogen, focusing on the scar maturation process. Frostbite is typically less prone to abnormal scarring compared to burns, and this report contrasts the differences in scar maturation between the two. A 31-year-old male hospital employee sustained first- to second-degree frostbite on his gloved hands from a 20-second exposure to liquid nitrogen while changing a cylinder. Conservative treatment was applied, and the patient was monitored for 9 months. The deeply affected area took 50 days to epithelialize but healed without hypertrophic scarring. A mild extension contracture was noted in the distal interphalangeal joint of the right index finger, but the skin remained supple and soft. Incidents of liquid nitrogen-induced frostbite are uncommon, with only 14 cases reported in PubMed® previously. In frostbite, the wound healing involves a slow replacement of damaged connective tissue, which acts as an internal splint, reducing wound contraction. This contrasts with burns, where rapid connective tissue replacement occurs, often leading to significant wound contraction due to the presence of myofibroblasts in granulation tissue. In the presented case, the slow healing process and minimal wound contraction led to mature scarring without abnormalities, underlining a distinctive healing trajectory in frostbite injuries compared to burns., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Burn Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Osteosarcoma treatment with limb-sparing surgery using liquid nitrogen can be applied to canine patients experiencing diminished quality of life after leg amputation. In particular, forelimb amputation may affect gait more than hindlimb amputation. In this study, limb-sparing surgery using liquid nitrogen was applied to primary osteosarcomas arising in the proximal scapula of a Welsh Corgi, the proximal humerus of a Golden Retriever, and the distal radius of a Great Pyrenees, according to the protocol of Tsuchiya et al. In all cases, postoperative radiographic examination revealed bone union between the treated and matrix bones. All patients recovered their gait postoperatively. These results suggest that limb-sparing surgery using liquid nitrogen-treated autologous bone is an effective option for patients with osteosarcoma.

This study was conducted in two steps to evaluate the influence of freezing methods and natural extracts on cryopreserved ram sperm quality. Initially, the research compared the effects of two freezing methods: liquid nitrogen (LN2) versus -80 °C, on post-thawed ram semen on total and progressive motilities and velocity parameters. Experiment I revealed no significant differences (P > 0.05) between the LN2 and -80 °C freezing methods, indicating similar effects on the analyzed parameters. Experiment II aimed to examine the influence of Spirulina platensis (SP) and Salvia verbenaca (SV) extracts added to egg yolk extender on cryopreserved sperm quality, utilizing the -80 °C freezing method. Various concentrations (1.25, 3.75, 6.25 and 8.75 µg*mL-1) of acetone (Ac-SP and Ac-SV) and hexanoic (Hex-SP), as well as methanolic (MeOH-SV) extracts, were added into the extender. A thorough assessment of post-thawed sperm quality parameters, encompassing motility, velocity parameters, viability, membrane integrity, abnormality and lipid peroxidation was conducted. The outcomes demonstrated that 1.25 and 3.75 g*mL-1 of Ac-SP and Hex-SP and 1.25 µg*mL-1 of AC-SV and MeOH-SV increased the post-thawed ram sperm quality. In conclusion, this study emphasizes the antioxidant properties of SP and SV extracts, highlighting their potential to protect cryopreserved sperm cells from oxidative stress at -80 °C.

Clean fire extinguishing systems applicable to the pottery jar liquor warehouse are in demand. In this study, taking 53vol% liquor as the research subject, fire models of various clean fire extinguishing systems comprising water mist, liquid carbon dioxide (LCO 2 ) and liquid nitrogen (LN 2 ) were established using a fire dynamic simulator to determine their fire extinguishing effect. A feasibility assessment of systems was performed under different fire source types, fire source sizes, and ventilation conditions. The fire extinguishing efficiency was analyzed in terms of the fire extinguishing time, oxygen concentration, and space temperature. The results showed that the success rate of the LCO 2 and LN 2 fire extinguishing systems was 100%, whereas the success rate of the water mist fire extinguishing system was 95%. In terms of reducing the oxygen concentration at the bottom of the space and the temperature in the space, the LCO 2 system exhibited the best performance, followed by the LN 2 system, and lastly the water mist. Under different ventilation conditions and fire source types, the LCO 2 fire extinguishing system was least affected, whereas the effectiveness of the water mist fire extinguishing system reduced under natural ventilation conditions, and the extinguishing efficiency of the LN 2 fire extinguishing system was affected by the fire source type. Overall, the LCO 2 system presented more advantages in extinguishing fires in pottery jar liquor warehouses and can provide a new idea for the development and application of clean and efficient fire extinguishing systems., (© 2024. The Author(s).)

This study examined the effects of liquid nitrogen vapor on osteogenesis in the rabbit femur. Cryotweezers made of porous nickel titanium alloy (nitinol or NiTi) obtained by self-propagating high temperature synthesis were used in this experiment. The porous structure of the cryotweezers allows them to hold up to 10 g of liquid nitrogen after being immersed for 2 min, which completely evaporates after 160 s. To study the effects of liquid nitrogen evaporation on osteogenesis, a rabbit femur was perforated. The formed holes were subjected to cryotherapy with varying exposure times. It was found that a 3 s exposure time stimulates osteogenesis, which was manifested in a greater number of osteoblasts in the regenerate compared to the control sample without liquid nitrogen. It was observed that increasing the exposure to 6, 9 or 12 s had a destructive effect, to varying degrees. The most severe damage was exerted by a 12 s exposure, which resulted in the formation of osteonecrosis areas. In the samples exposed to 6 and 9 s of cryotherapy, destruction of the cytoplasm of osteocytes and osteoclasts was observed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

The structure and properties of materials are determined by a diverse range of chemical bond formation and breaking mechanisms, which greatly motivates the development of selectively controlling the chemical bonds in order to achieve materials with specific characteristics. Here, an orientational intervening bond-breaking strategy is demonstrated for synthesizing ultrathin metal-organic framework (MOF) nanosheets through balancing the process of thermal decomposition and liquid nitrogen exfoliation. In such approach, proper thermal treatment can weaken the interlayer bond while maintaining the stability of the intralayer bond in the layered MOFs. And the following liquid nitrogen treatment results in significant deformation and stress in the layered MOFs' structure due to the instant temperature drop and drastic expansion of liquid N 2 , leading to the curling, detachment, and separation of the MOF layers. The produced MOF nanosheets with five cycles of treatment are primarily composed of nanosheets that are less than 10 nm in thickness. The MOF nanosheets exhibit enhanced catalytic performance in oxygen evolution reactions owing to the ultrathin thickness without capping agents which provide improved charge transfer efficiency and dense exposed active sites. This strategy underscores the significance of orientational intervention in chemical bonds to engineer innovative materials., (© 2024 Wiley‐VCH GmbH.)

BACKGROUND QUERCUS: seeds that are recalcitrant to desiccation and freezing temperatures cannot be stored in gene banks under conventional conditions. However, the germplasm of some recalcitrant seeded species can be stored in liquid nitrogen (-196 °C). Unfortunately, for many species, among them for almost the whole genus Quercus, an effective cryostorage method is still unknown. In this study, we propose a successful cryostorage protocol for Quercus petraea (Matt.) Liebl. germplasm using plumules (a shoot apical meristem of an embryo) frozen on aluminium cryo-plates. RESULTS: The plumules isolated from the acorns of ten provenances were prestored in 0.5 M sucrose solution (for 18 h). To form alginate beads (one plumule per bead), the plumules were placed in the wells of a cryo-plate and embedded in calcium alginate gel. For cryoprotection, the encapsulated plumules were immersed in cryoprotectant solution containing 2.0 M glycerol and different concentrations of sucrose (0.8-1.2 M) for 40 min at 25 °C and desiccated under a laminar flow cabinet for 1.0-4.0 h. Cryo-plates with plumules were directly immersed in liquid nitrogen and then cryostored for 30 min. For rewarming, cryo-plates with plumules were immersed in 1.0 M sucrose solution and rehydrated for 15 min at 25 °C. Survival rates varied from 25.8 to 83.4 were achieved after cryoprotection in 1.0 M sucrose solution and the drying of plumules for 2 h. The in vitro regrowth rate of cryopreserved plumules varied among provenances and was 26-77%. CONCLUSIONS: This study presents, for the first time, a successful, simple and effective protocol for the cryopreservation of Q. petraea germplasm that could be used in gene banks. The experiment was successfully repeated on seeds from various provenances, each yielding similar, good results. However, seed quality and storage time after harvesting are important factors in plumule regrowth after cryopreservation., (© 2024. The Author(s).)

Background/aim: Tumor-induced osteomalacia (TIO) is a rare pathology caused by overproduction of fibroblast growth factor 23 (FGF23). Its common clinical features include generalized muscle weakness, bone pain, and fractures. Complete resection of the offending tumor is the mainstay treatment. In this report, we present the first case of TIO by an FGF23 producing tumor treated using a tumor-bearing autograft treated with liquid nitrogen., Case Report: A 63-year old female presented with generalized body pain, particularly in the left arm. The patient was diagnosed with a FGF23 producing tumor of the left humerus. Wide resection of the involved tumor was performed using a tumor-bearing autograft that was treated with liquid nitrogen. Postoperatively, the FGF23 and alkaline phosphatase (ALP) levels significantly decreased and inorganic phosphate normalized. There was also subsequent relief of generalized body pain. Immediately after the operation, range of motion of the left shoulder and elbow was initiated. The patient was instructed to perform forward flexion and abduction up to 90° with a rotational restraint. Almost complete bone union was observed at 12 months post procedure. Postoperative functional results were as follows: Musculoskeletal Tumor Society (MSTS) score of 27/30, 90% and International Society of Limb Salvage (ISOLS) score of 26/30, 87%. Ten years after the surgery, osteotomy line was completely obscured based on radiographs. The patient was disease free and without activity limitation., Conclusion: This is the first case report of wide excision of a FGF23 producing tumor and reconstruction using a tumor-bearing frozen autograft performed with excellent outcomes., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Bone grafting has emerged as a key solution in bone defect management such as allograft, graft of bone from another individual. However, bone allografts usually undergo rigorous preparation to eliminate immune-triggering elements. The deep-freezing methods may delay graft use, while cryopreservation using liquid nitrogen allows rapid freezing but may alter graft characteristics. The aim of this study was to investigate the post-preservation changes in bone allograft characteristics and to compare the effectiveness of deep-freezing and liquid nitrogen methods using animal model. An experimental study using a post-test only control group design was conducted. Fresh-frozen femoral cortical bone was obtained from male New Zealand white rabbits. Preservation by deep-freezing involved placing bone samples in a -80°C freezer for 30 days. For liquid nitrogen preservation, bone grafts were immersed in liquid nitrogen for 20 min, followed by a 15-min rest at room temperature and a final immersion in 0.9% sodium chloride at 30°C for 15 min. Bone samples then underwent evaluation of cell viability, compression, and bending tests. Cell viability test employed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the compression and bending tests used the Universal Testing Machine (UTM). Independent Student t-test or Mann-Whitney U test were used to compare the methods as appropriate. Our study found that the use of deep-freezing and liquid nitrogen resulted in similar outcomes for cell viability, compression, and bending tests, with p -values of 0.302, 0.745, and 0.512, respectively. Further exploration with larger sample sizes may help to optimize the methods for specific applications., Competing Interests: All the authors declare that there are no conflicts of interest., (© 2024 by the authors.)

Coolant-assisted liquid nitrogen (LN) flash freezing of frozen tissues has been widely adopted to preserve tissue morphology for histopathological annotations in mass spectrometry-based spatial proteomics techniques. However, existing coolants pose health risks upon inhalation and are expensive. To overcome this challenge, we present our pilot study by introducing the EtOH-LN workflow, which demonstrates the feasibility of using 95 % ethanol as a safer and easily accessible alternative to existing coolants for LN-based cryoembedding of frozen tissues. Our study reveals that both the EtOH-LN and LN-only cryoembedding workflows exhibit significantly reduced freezing artifacts compared to cryoembedding in cryostat (p < 0.005), while EtOH-LN (SD = 0.56) generates more consistent results compared to LN-only (SD = 1.29). We have modified a previously reported morphology restoration method to incorporate the EtOH-LN workflow, which successfully restored the tissue architecture from freezing artifacts (p < 0.05). Additional studies are required to validate the impact of the EtOH-LN workflow on the molecular profiles of tissues., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest to report., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The frozen inactivation of autologous tumor bones using liquid nitrogen is an important surgical method for limb salvage in patients with sarcoma. At present, there are few research reports related to frozen inactivated autograft replantation., Methods: In this study, we retrospectively collected the clinical data of patients with bone and soft tissue sarcoma treated with liquid nitrogen-frozen inactivated tumor bone replantation, and analyzed the safety and efficacy of this surgical method. The healing status of the frozen inactivated autografts was evaluated using the International Society of Limb Salvage (ISOLS) scoring system. Functional status of patients was assessed using the Musculoskeletal Tumor Society (MSTS) scale., Results: This study included 43 patients. The average length of the bone defect after tumor resection is 16.9 cm (range 6.3-35.3 cm). Patients with autograft not including the knee joint surface had significantly better healing outcomes (ISOLS scores) (80.6% ± 15% vs 28.2% ± 4.9%, P<0.001) and limb function (MSTS score) (87% ± 11.6% vs 27.2% ± 4.4%, P<0.001) than patients with autografts including the knee joint surface. The healing time of the end of inactivated autografts near the metaphyseal was significantly shorter than that of the end far away from the metaphyseal (9.8 ± 6.3 months vs 14.9 ± 6.3 months, P=0.0149). One patient had local recurrence, one had an autograft infection, five (all of whom had an autograft including the knee joint surface) had joint deformities, and seven had bone non-union., Conclusion: Frozen inactivated autologous tumor bone replantation is safe and results in good bone healing. But this method is not suitable for patients with autograft involving the knee joint surface., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tian, Dong, Yang, Qu, Liu, Liu, Ma, Wang and Yao.)

Introduction: The recommended treatment method for bone sarcoma is wide local excision and reconstruction to preserve limb function. Established methods of reconstruction are mega prosthesis or biological reconstruction. This study aimed to determine the complications and functional outcomes associated with limb salvage surgery using liquid nitrogen-treated bone., Materials and Methods: We retrospectively observed the short-term outcome of limb salvage surgeries where liquid nitrogen bone was used for reconstruction. A total of 15 patients underwent reconstruction with liquid nitrogen auto graft from January 2018 to December 2020. We used the free freezing method of liquid nitrogen treatment after wide local excision of sarcoma. We observed short-term outcomes after liquid nitrogen-treated bone reconstruction in limb salvage surgery. Survival of the auto grafts was recorded using the Kaplan-Meier method with a 95% confidence interval., Results: The mean follow-up was 19.83 ± 4.5 months. The mean musculoskeletal tumor society score was 62.4 ± 7.9%, while the average Toronto extremity score was 59.6 ± 5.7%. Three patients died during the study duration due to visceral metastasis. Skin necrosis and wound breakdown were major complications in 9 (60%) patients. Deep infection was observed in 4 (26.7%). Similarly, 4 (26.7%) patients had non-union at either the proximal or distal osteotomy site, while the average time of bone union in the rest of the patients was 6.3 ± 1.7 months. A total of 6 (40%) patients underwent reoperation after liquid nitrogen treatment, either due to infection or non-union at the osteotomy site. Recurrence was observed in 3 (20%) of patients., Conclusion: We observed a high complication rate with liquid nitrogen-treated autograft reconstruction. Vascularized fibula with liquid nitrogen-treated autograft or endoprosthesis should be encouraged., Competing Interests: The author(s) declare(s) that there is no conflict of interest., (Copyright: © 2024 Farooq MZ, et al.)

Cryopreservation, or the storage at liquid nitrogen temperatures (-196°C), of embryogenic cells or somatic embryos allows their long-term conservation without loss of their embryogenic capacity. During the last decade, protocols for cryopreservation of embryogenic material of woody species have been increasing in number and importance. However, despite the large experimental evidence proved in thousands of embryogenic lines, the application for the large-scale conservation of embryogenic material in cryobanks is still limited. Cryopreservation facilitates the management of embryogenic lines, reducing costs and time spent on their maintenance, thus limiting the risk of the appearance of somaclonal variation or contamination. Somatic embryogenesis in combination with cryopreservation is especially useful to preserve the juvenility of lines while the corresponding clones are being field-tested. Hence, when tree performance has been evaluated, selected varieties can be propagated from the cryostock. The traditional method of slow cooling or techniques based on vitrification are mostly applied procedures. For example, slow cooling methods are widely applied to conserve embryogenic lines of conifers. Desiccation based procedures, although simpler, have been applied in a smaller number of species. Genetic stability of the cryopreserved material is supported by multiloci PCR-derived markers in most of the assayed species, whereas DNA methylation status assays showed that cryopreservation might induce some changes that were also observed after prolonged subculture of the embryogenic lines. This article reviews the cryopreservation of embryogenic cultures in conifers, fruit species, deciduous forest species and palms, including a description of the different cryopreservation procedures and the analysis of their genetic stability after storage in liquid nitrogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ballesteros, Martínez, Sánchez-Romero, Montalbán, Sales, Moncaleán, Arrillaga and Corredoira.)

Healthy insect cell cultures are critical for any method described in this book, including making productive baculovirus banks, protein or AAV expression, and determining viral titers. This chapter describes cell maintenance in shake flasks using serum-free conditions and the expansion of virus stocks from a single plaque purified virus. Insect cells can be passaged over multiple generations, but as the cells may undergo changes over multiple passages, limiting the use of your cells to a defined number of passages such as 50 passages is recommendable. Baculovirus stocks once created using serum-free media are not very stable at 4-8 °C. This chapter also includes a simple method to store cells from an early cell passage and your virus stock in liquid nitrogen., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Transcriptomic studies often require collection of fresh tissues post euthanasia. The chosen euthanasia method might have the potential to induce variations in gene expressions that are unlinked with the experimental design. The present study compared the suitability of 'nitrogen gas in foam' (ANOXIA) in comparison to a non-barbiturate anaesthetic, T-61® (T61), for euthanizing piglets used in transcriptome research. Further, the effect of common tissue storage conditions, RNAlater™ (RL) and snap freezing in liquid nitrogen (LN 2 ), on gene expression profiles were also analysed., Results: On comparison of the 3'mRNA-Seq data generated from pituitary, hypothalamus, liver and lung tissues, no significant differential expression in the protein coding genes were detected between the euthanasia methods. This implies that the nitrogen anoxia method could be a suitable alternative for euthanasia of piglets used in transcriptomic research. However, small nuclear RNAs (snRNAs) that constitute the eukaryotic spliceosomal machinery were found to be significantly higher (log2fold change ≥ 2.0, and adjusted p value ≤ 0.1) in pituitary samples collected using ANOXIA. Non-protein coding genes like snRNAs that play an important role in pre-mRNA splicing can subsequently modify gene expression. Storage in RL was found to be superior in preserving RNA compared to LN 2 storage, as evidenced by the significantly higher RIN values in representative samples. However, storage in RL as opposed to LN 2 , also influenced differential gene expression in multiple tissues, perhaps as a result of its inability to inhibit biological activity during storage. Hence such external sources of variations should be carefully considered before arriving at research conclusions., Conclusions: Source of biological variations like euthanasia method and storage condition can confound research findings. Even if we are unable to prevent the effect of these external factors, it will be useful to identify the impact of these variables on the parameter under observation and thereby prevent misinterpretation of our results., (© 2023. The Author(s).)

Purpose: To describe a simple cryosurgical technique for treating canine distichiasis, its success rate and any associated complications., Methods: Clinical records from canine patients undergoing treatment for distichiasis using a specific cryotherapy technique were retrospectively reviewed. The technique employed a triple freeze-thaw cycle via a transconjunctivally applied, liquid nitrogen-cooled, 2-mm diameter closed cryoprobe., Results: The cryotherapy technique was employed in 125 dogs with distichiasis over a period of 13 years, with 109 (87%) bilaterally and 16 (13%) unilaterally affected, totaling 234 eyes. The mean age at diagnosis was 1.9 years (4 months to 7.5 years), with Staffordshire bull terriers the most common breed (31%). Treatment was deemed successful if clinical signs related to distichiasis resolved, including blepharospasm, conjunctival hyperemia and excess lacrimation. A single triple-freeze cryotherapy treatment eliminated distichiae in 195/234 (83%) eyes at a mean follow-up of 4 months (2 weeks to 10 years). Minor recurrence of distichiae in previously treated regions was observed in 39/234 (17%) eyes. Repeat cryotherapy was required in 25/234 (11%) eyes, with the remaining eyes asymptomatic. The two most common postoperative complications were mild to moderate eyelid swelling in all eyes immediately following surgery and eyelid margin depigmentation in 59/234 eyes (25%) between two and 4 weeks postoperatively, both of which were temporary., Conclusion: This cryotherapy technique for treating canine distichiasis is relatively simple and safe to perform, has a high success rate and a low recorded complication rate, but would benefit from further assessment of any potential detrimental effects on qualitative tear film components., (© 2023 American College of Veterinary Ophthalmologists.)

In microbiome studies, fecal and oral samples are stored and processed in different ways, which could affect the observed microbiome composition. In this study, we compared storage and processing methods applied to samples prior to DNA extraction to determine how each affected microbial community diversity as assessed by 16S rRNA gene sequencing. We collected dental swabs, saliva, and fecal samples from 10 individuals, with three technical replicates per condition. We assessed four methods of storing and processing fecal samples prior to DNA extraction. We also compared different fractions of thawed saliva and dental samples to fresh samples. We found that lyophilized fecal samples, fresh whole saliva samples, and the supernatant fraction of thawed dental samples had the highest levels of alpha diversity. The supernatant fraction of thawed saliva samples had the second highest evenness compared to fresh saliva samples. Then, we investigated the differences in observed community composition at the domain and phylum levels and identified the amplicon sequence variants (ASVs) that significantly differed in relative abundance between the conditions. Lyophilized fecal samples had a greater prevalence of Archaea as well as a greater ratio of Firmicutes to Bacteroidetes compared to the other conditions. Our results provide practical considerations not only for the selection of storage and processing methods but also for comparing results across studies. Differences in processing and storage methods could be a confounding factor influencing the presence, absence, or differential abundance of microbes reported in conflicting studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Superdock, Zhang and Poole.)

This paper describes a procedure for the validation of alpha-particle sources (exempt unsealed sources) to be used in experimental setups with liquefied gases at cryogenic temperatures (down to -196 °C) and high vacuum. These setups are of interest for the development and characterization of neutrino and dark matter detectors based on liquid argon, among others. Due to the high purity requirements, the sources have to withstand high vacuum and cryogenic temperatures for extended periods. The validation procedure has been applied to 241 Am sources produced by electrodeposition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: Extremity-sparing surgery should be the main objective if a functional extremity is to be obtained in cases of malignant bone tumors. After extensive resection, numerous techniques have been described to reconstruct bone defects. This study aimed to compare the outcomes of patients who underwent external radiation therapy and liquid nitrogen biological reconstruction at two different facilities., Methods: The study included 12 patients who received biological reconstruction therapy for bone sarcoma and had at least two years of follow-up. Demographic data, pathological diagnosis, presence of systemic metastasis, and recurrence during follow-up were among the information logged. Patients who used liquid nitrogen were placed in group 1, and those who underwent external irradiation were placed in group 2. After being contacted for their final follow-up appointments, the outcomes were compared by recording the Musculoskeletal Tumor Society Score (MSTSS)., Results: For participants with a mean age of 10.75±3.6 (5-17), the follow-up period was 30.2±16.3 months in total. In contrast to the patients in group 1, who experienced union on average after 7.5±1.2 months, those in group 2 experienced union after 7.6±1.1 months. Patients in group 1 had an MSTSS of 75.5±11.8%, while those in group 2 had a score of 77±4.4. There was no discernible difference between the two groups' union times (p>0.05). There was no statistically significant difference between the two groups' MSTSS (p>0.05)., Conclusion: After tumor resection, extracorporeal radiation therapy and the application of liquid nitrogen are still useful treatment options and neither of them is superior to the other., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Emet et al.)

Background: Cryoballoon ablation (CBA) has emerged as an effective treatment for atrial fibrillation (AF)., Objectives: This study sought to assess the performance of a novel liquid nitrogen-driven CBA system and evaluate its safety and efficacy in the treatment of drug-resistant paroxysmal atrial fibrillation (PAF)., Methods: This was a prospective multicenter single-arm clinical trial with 10 participating tertiary hospitals enrolling 176 patients with PAF. All participants received liquid nitrogen-driven CBA developed by the Cryofocus Medtech Company. Scheduled follow-up was performed before discharge and 3 months, 6 months, and 12 months after CBA. The primary endpoints were defined as 1) treatment success (freedom from antiarrhythmic drugs and atrial tachycardia at 12 months after CBA); and 2) immediate success rate of pulmonary vein isolation. The safety endpoint was the incidence of device- and procedure-related adverse events (AEs) and all-cause mortality., Results: A total of 172 participants were included, with an average age of 59.22 ± 9.25 years and 99 (57.56%) of them men. Immediate success rate was 97.67% (95% CI: 94.15%-99.36%) and 12-month treatment success rate was 82.56% (95% CI: 76.89%-88.23%), including a late recurrence rate of 13.61%. Incidences of device- and procedure-related AEs were 2.27% and 25.00%, respectively. Phrenic nerve palsy (PNP) occurred in 6 patients, of which 5 recovered during follow-up. Although the incidence of total severe AEs was 17.05%, including an all-cause mortality of 0.57%, only 1 case of permanent PNP was related to the CBA procedure., Conclusions: This premarketing prospective multicenter single-arm clinical trial demonstrated that the liquid nitrogen cryoablation system is safe and effective in the treatment of PAF., Competing Interests: This study was supported by Shanghai Hospital Development Center, Shanghai, China (grant no. SHDC2020CR1040B). The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

Nanomaterials with unique structural and properties can be synthesized by rapid transition of the thermodynamic state. One promising method is through electrical explosion, which possesses ultrafast heating/quenching rates (d T /d t ~10 9 K/s) of the exploding conductor. In this study, experiments were performed with fine metallic wire exploding in liquid nitrogen (liq N2, 77 K) under different applied voltages. For the first time in the literature, the physical image of the electrical explosion dynamics in liq N2 is depicted using electro-physical diagnostics and spatial-temporal-resolved photography. Specifically, the pulsation and collapse processes of the vapor bubble (explosion products) have been carefully observed and analyzed. As a comparison, an underwater electrical explosion was also performed. The experimental results suggest that the vapor bubble behavior in liq N2 differs from that in water, especially in the collapse phase, characterized by secondary small-scale bubbles in liq N2, but multiple bubble pulses in water; correspondingly, the products' characteristics are discrepant.

Purpose: The demand for fertility preservation continues to grow as cancer treatment outcomes improve. The specimen storage period is longer for fertility preservation than for conventional fertility treatment; therefore, a robust management system for stored specimens is required. We conducted the first national survey in Japan on the management of cryopreserved specimens in fertility preservation facilities. Methods: Questionnaires were mailed to 130 fertility preservation facilities. Primary outcomes included the official position of the storage manager, support system in case of facility closure, disaster countermeasures, management and operating system for liquid nitrogen storage containers, preservation costs, and method to confirm the intention to continue storage. Results: The response rate was 63.8%. The facility director most commonly functioned as the storage manager (59.0%). In case of facility closure, 20.5% had an approved transfer site; 59.0% had not made any decisions. In the management of liquid nitrogen containers, 83.1% regularly replenished the liquid nitrogen, 65.1% regularly checked the amount, and 16.9% had alarm monitoring systems. Regarding disaster countermeasures, 70.9% had taken measures to protect specimens. Conclusion: This survey revealed issues such as disparities among facilities regarding long-term specimen storage systems. Accordingly, management standards for fertility preservation facilities should be established.

Potatoes are consumed by millions of people and are the survival food in several countries. Cultivated varieties of potato (Solanum tubersosum L.) are results of selection and crossing of many wild species. Only 8-13% of wild potato species used for food are preserved by either in situ or ex situ methods. The U.S. National Potato Germplasm Collection maintains over 5900 accessions, of which 75% are crop wild relatives (CWR). The objective of the study was to investigate regrowth of cryogenically stored clonal propagules (shoot tips) of selected CWR accessions maintained in the collection. Sixty-nine accessions from 30 Solanum species and six accessions that are not yet assigned to a species were cryopreserved by a droplet vitrification method at the NLGRP. The post cryopreservation regrowth varied from 40 to 100% (average 68%) but was not significantly different between the tested accessions. Regrowth of six accessions tested after 10 years of cryogenic storage was between 35 and 90% (average 66%) and was significantly different from their initial regrowth (average 87%); the largest viability loss was in S. condolleanum; but for the other five accessions the regrowth was between 45 and 90% (average 72%) and suggested at least 10 years of successful storage in LN was possible. Twelve potato wild species cryopreserved in this study were reported in literature as important for developing cultivated varieties for changed weather conditions., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Chondrosarcoma is the second most common sarcoma of bone, accounting for about 25% of primary bone tumors. Histological grading is the single most important determinant of local recurrence and metastases, where low-grade chondrosarcoma (grade 1: atypical cartilaginous lesion) is associated with 10 years survival rate 79-100%. There has been always a debate about the need for wide resection in managing low-grade chondrosarcoma, where several reports describe successful management with intra-lesional curettage and adjuvant therapy, minimizing the need for complex reconstructive procedures and improving the expected outcomes., Hypothesis: Grade 1 chondrosarcoma of the appendicular skeleton can be successfully treated through extended curettage and liquid nitrogen with favorable oncological and functional outcomes., Patients and Methods: We retrospectively assessed 113 patients with low grade chondrosarcoma in the appendicular skeleton, who were treated by extended curettage, adjuvant therapy using liquid nitrogen, reconstruction using polymethyl methacrylate and prophylactic fixation. Patients were assessed regarding incidence of recurrence, incidence of non-oncological complications and functional outcome., Results: The mean follow up period was 110 months. In three patients (2.7%), local recurrence occurred. Five point three percent of the patients got non-oncological complications that were successfully managed. The mean MSTS score was 95%, with no evidence of distant metastases or disease related mortality., Conclusion: Grade 1 chondrosarcoma in the appendicular skeleton can be effectively treated using extended curettage, liquid nitrogen as adjuvant and cement as filler. Prophylactic fixation is required to avoid pathological fractures. When done properly, excellent functional outcome is expected., Level of Evidence: IV, Case series (retrospective comparative study)., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

One major goal of biobanks is to provide the best possible biospecimen quality for research use. This can be achieved, notably in accredited structures, by using standardized procedures for collection, processing, and storage of biosamples and associated data. Since tissue samples of a clinical biobank are commonly collected at surgical theaters in satellite locations or hospitals in remote areas, adequate temporary storage of the biosample is mandatory to maintain optimal sample quality. In cases where immediate snap freezing of the collected material is possible, interim storage of the samples in portable dewars filled with liquid nitrogen (LN 2 ) is a widely used method. Therefore, the ideal dewar size and maximum storage time need to be considered to maintain an optimal biospecimen quality. In addition, the nature of the cryotube material is an important aspect for keeping the biosample safe while storing it in LN 2 . The objective of this study was to test different dewar vessels with respect to LN 2 volume and consumption and to analyze the impact of LN 2 contact on cryotube material through scanning electron microscopy.

The utilization of T cells is becoming increasingly prominent in both clinical and research domains. However, the need to optimize preservation methodologies for extended periods of time remains unmet. To address this issue, we have developed a protocol for the handling and preservation of T cells that facilitates successful donor homologous co-cultures with dendritic cells (DCs), and preserves the cells for subsequent testing. Our method enhances experimental efficiency by reducing time and effort, and simplifying the use of T cells in mono or co-cultures. Our T cell handling and preservation methodology demonstrates the stability and viability of these cells in co-cultures, with viability exceeding 93% before and after liquid nitrogen preservation. Additionally, the preserved cells display no unspecific activation, as evidenced by the unaltered expression of the T cell activation marker CD25. The proliferation profile of preserved T cells used in DC-T cell co-cultures, stimulated by lipopolysaccharide (LPS)-activated DCs, attests to the potency and ability of these cells to interact and proliferate. These findings underscore the efficacy of our handling and preservation methodology in maintaining T cell viability and stability. Preserving donor T cells not only reduces the inconvenience of repeated blood donations but also enhances accessibility to a particular population of T cells for experimental or clinical applications, such as chimeric antigen receptor T cells., Competing Interests: Conflict of interests The authors have no conflict of interests to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Zero-valent manganese (ZVMn) possesses high reducibility in theory, while sulfide exhibits strong affinity towards a variety of heavy metals owing to the low solubility of metal sulfides. Yet the performance and mechanisms on using sulfidized zero-valent manganese (SZVMn) to remove thallium (Tl) from wastewater still remain unclear. In this study, the performance of Tl(I) removal using SZVMn synthesized by borohydrides reduction followed by sulfides modification, with and without liquid nitrogen treatment, was compared and the mechanism behind was investigated. The results show that at a S/Mn molar ratio of 1.0, liquid nitrogen modified SZVMn (LSZVMn) possessed more interior channels and pores than SZVMn, with 65.3% higher specific surface area and 73.7% higher porosity, leading to 6.4-8.1% improvement in adsorption of Tl(I) at pH 4-10. LSZVMn showed effectiveness and robustness in Tl(I) removal in the presence of co-existing ions up to 0.1 M. The adsorption of Tl(I) conformed to the pseudo-1st-order kinetic model, and followed the Langmuir isothermal model, with the maximum Tl adsorption capacity of 264.9 mg·g -1 at 288 K. The mechanism of Tl(I) removal with SZVMn was found to include sulfidation-induced precipitation, manganese reduction, surface complexation, and electrostatic attraction. The liquid nitrogen pretreatment embrittled and cracked the outer shell of S/Mn compounds, resulted in a highly hierarchical structure, enhancing the manganese reduction and improving the Tl(I) removal. Based on the above results, the SZVMn and its liquid nitrogen-modified derivatives are novel and effective environmental materials for Tl(I) removal from wastewater, and the application of SZVMn to the removal of other pollutants merits investigation in future study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Butternut (Juglans cinerea L.), a species listed as endangered, is currently undergoing rapid decline due to habitat loss and the introduction of Ophiognomonia clavigignenti-juglandacearum, a non-native pathogen causing butternut canker. The decline of butternut has led to the development of cryobiological methods for the ex-situ conservation of the species since viability is not maintained using conventional methods. In this study, we assess the survivability and growth of butternut embryogenic axes into plantlets after 7 years of cryopreservation. Results show that cryopreservation is a highly effective method for the long-term storage of embryogenic axes since both survival and subsequent acclimatization into plantlets was successful with an average of 76% survival and 71% acclimatization, respectively. These results surpass the actual duration viability for non-cryogenic storage of butternut seed and we hope that future testing will result in longer term success of this cryobiological method and provide much needed germplasm for future restoration., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2023. Published by Elsevier Inc.)

Animals used in research often have to be euthanised, especially when tissue sampling is essential. Recently, a euthanasia method, utilizing an inhalant anaesthetic 'nitrogen gas in foam' in an anoxia box (ANOXIA TM ), has gained considerable interest as it claimed to be more animal-friendly. However, it is not clear whether the use of this euthanasia method has an influence on RNA measurements. Moreover, there are no studies on the interaction effect of different euthanasia methods on the tissue sample storage conditions. The current study compared RNA measurements from two euthanasia methods (ANOXIA TM vs. T-61 ® injection) and two storage conditions (RNA later ™ vs. snap freezing) in 12 male piglets. The nitrogen anoxia method had a significantly higher RNA yield ( p < 0.01) compared to the T-61 ® method. However, no effect of the euthanasia methods on the A260/230 ratio and RIN value was observed. Tissues stored in RNA later ™ had significantly higher RIN values ( p < 0.001) compared to snap frozen samples. The present study could not find a significant difference between the two euthanasia methods in piglets, with regard to RNA quality measurements. Hence, the nitrogen anoxia technique (ANOXIA TM ) might be considered as a suitable alternative to T-61 ® for euthanasia of piglets used in research.

A unique and valuable diversity of the Malus and Pyrus wild species germplasm is maintained ex situ in field collections in the 'Fruit Genebank' in Dresden-Pillnitz, Germany. (1) Background: The establishment of a duplicate collection is necessary to preserve this material safely from abiotic and biotic stress factors. (2) Methods: Two different techniques, cryopreservation using dormant buds and PVS2 vitrification using in vitro shoot tips, were applied and compared. (3) Results: In Malus altogether 180 accessions belonging to 32 species were processed with an average recovery rate of 39% within ten years using the dormant bud method. Accessions, 116 in number, achieved the criterion of 40% recovery which was 64.44% of all accessions tested. In the case of Pyrus germplasm a total of 35 accessions of 21 species and both techniques for cryopreservation were tested. In the results of these experiments the PVS2 method led to better results compared to the dormant bud method. (4) Conclusions: In Malus the dormant bud technique will be the method of choice for the future to build up the duplicate collection. Further experiments using both techniques are planned in the future to make a final decision for Pyrus .

Histological and molecular genetic evaluation of skeletal and cardiac muscles is an indispensable part of understanding muscle biology and the pathology of muscle disorders. Proper processing of the muscle tissue is a prerequisite for optimal evaluation. However, the processing of skeletal muscle samples often comes with many challenges. One of the commonly used methods of frozen tissue preparation involves optimal cutting temperature compound (OCT compound) embedding. This method is considered optimal for the processing of most of the routinely studied tissue samples. However, the processing of skeletal muscle samples using this method is often unsuitable as it causes artifacts and low DNA, RNA, and protein yield and quality due to the slow freezing of skeletal muscle tissues that allows ice crystals to form. One of the most suitable methods for skeletal muscle tissue processing for histological, genetic, and molecular studies is rapid freezing of freshly collected tissue samples using isopentane cooled with liquid nitrogen and tragacanth gum, which provides distinct advantages in consuming less time, preserving the cell morphology, and helping higher nucleic acids and protein yields. This chapter describes a protocol for rapid freezing of freshly collected skeletal muscle tissues using isopentane pre-chilled with liquid nitrogen and tragacanth gum. Skeletal muscle tissue samples processed using this protocol can be used for histological and immunological staining investigations and studies requiring DNA, RNA, and proteins from these tissues., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lipids are a heterogeneous group of substances characterized by their solubility in organic solvents and insolubility in water. Lipids can be found as normal components of different tissues and organs, and they can be affected by several pathological conditions. The histochemical identification of lipids plays an important role in the histopathological diagnosis and research, but successful staining depends on adequate fixation and processing of the tissue. Here we describe methods to fix, cryoprotect, and process tissue samples for the histochemical identification of lipids in frozen or paraffin-embedded tissues., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Osteosarcoma is a common primary malignancy of the bone. Osteosarcoma prognosis improves tremendously when chemotherapy is given in adjunct to surgical intervention. Limb reconstruction with sterilized autologous bone graft stabilized by orthopaedics implants has become a choice of treatment, but the biomechanical property of the bone treated with such sterilization method is a point of concern as a graft with inferior biomechanical property might lead to graft failure. This study compares the biomechanical properties of the bones treated with cryotherapy using liquid nitrogen and pasteurization in the form of four-point bending compression tests. Six sheep tibia bones were used for each group of treatment with one control group where no treatment was given. In the four-point bending test, osteotomy was performed at the tibia; the proximal tibia was treated with liquid nitrogen and pasteurization respectively. The treated bone is then reconstructed with the corresponding distal bone with locking plate and screws (Jiangsu Trauhiu Medical Instrument Co. Ltd., Changzhou, China). The four-point bending test was performed with an Electropulse® E3000 universal testing machine (Instron®, High Wycombe, United Kingdom) and results in the form of maximum compressive load, stress, and strain were collected. Photographic analysis of the fracture location and pattern were documented and analysed. We found that both methods of sterilization did not alter the biomechanical properties of the treated bone to the extent of statistical significance in comparison with the control group. However, other factors such as histological changes post treatment, equipment availability, and long-term outcome must be considered., Competing Interests: The authors have declared financial relationships, which are detailed in the next section., (Copyright © 2022, Nik Abdul Adel et al.)

Background: Percutaneous cryoablation with liquid nitrogen is a new technique being used in the treatment of some malignant tumors. Our objective was to assess its feasibility in the ablation of tumor lesions of various sizes and locations., Methods: This retrospective, monocentric study included all consecutive patients who underwent percutaneous cryoablation with liquid nitrogen between December 2019 and March 2021. Cryoablation was performed using 10G or 13G cryoprobes. The ablation volume was measured on post-treatment CT or MRI., Results: 22 patients (24 lesions) were included, 16 of whom were men (73%), while median age was 66 years. The lesions were located in the bone (42%), kidney (29%), soft tissue (17%), lung (8%), or liver (4%). It was feasible in all tumor locations and produced median ablation zones 25 mm in width and 35 mm in length, with a 23 min median freezing time. Freezing duration was correlated with the ice volume ( p Spearman = 0.02), but not with the ablation volume ( p = 0.11). The average difference between the ablation zone and ice ball sizes were -6.4 mm in width and -7.7 mm in length. Both ice and ablation volumes were larger when using the 10G probe as compared to when the 13G was used. No complications were reported., Discussion: We showed that this technique was safe and feasible in all organs tested. The freezing duration was correlated with the ice ball size, but not with the ablation zone.

Introduction: Long-term spinal stability after total en bloc spondylectomy (TES) is challenging. The aim of this study was to examine whether the new method could reduce the incidence of instrumentation failure (IF)., Methods: We retrospectively compared 116 patients with spinal tumors who underwent TES between 2010 and 2019 and were followed up for >1 year. IF, cage subsidence, and complications were evaluated. Propensity score matching between conventional and new method groups was performed for age, sex, body mass index, preoperative radiotherapy, number of resected vertebrae, number of instrumented vertebrae, tumor level, and follow-up period. There were 25 cases each in the conventional and new method groups. The conventional method used a titanium mesh cage for anterior reconstruction and 5.5-mm-diameter titanium alloy rods for posterior fixation. The new method used a more robust cage for anterior reconstruction, bone grafting was performed around the cage, and 6.0-mm-diameter cobalt chromium rods were used for posterior fixation. We compared the incidence of IF and cage subsidence after TES between the conventional and new method groups., Results: While 5 out of 25 patients (20.0%) in the conventional method group experienced IF, none from the new method group experienced IF. Three-year implant survival rates were 87.3% in the conventional and 100% in the new method groups. The new method group had a significantly higher implant survival rate (p<0.01). Cage subsidence was observed in 11 of 25 (44/0%) patients in the conventional method and 1 of 25 (4.0%; significantly lower, p<0.05) in the new method group., Conclusions: The new reconstruction method significantly reduced IF incidence in patients with TES., Competing Interests: Conflicts of Interest: The authors declare that there are no relevant conflicts of interest., (Copyright © 2023 The Japanese Society for Spine Surgery and Related Research.)

Background: For patients with malignant limb tumors, salvage surgery can be achieved using endoprosthesis or biological reconstructions like allograft or autograft. In carefully selected patients, resected bone can be recycled after sterilization using methods like autoclaving, irradiation, pasteurization or freezing with liquid nitrogen. We evaluated the clinical outcome and complications of malignant limb tumors treated with intercalary resection and frozen autograft reconstruction., Methods: We reviewed 33 patients whose malignant bone tumors were treated by wide resection and reconstruction with recycling liquid nitrogen-treated autografts between 2006 and 2017. Limb function, bone union at the osteotomy site and complications were evaluated. Functional outcome was assessed using the Musculoskeletal Tumor Society (MSTS) scoring system., Results: The cohort comprised 16 males and 17 females, with a mean age of 35.4 years (14-76 years). The most common tumor was osteosarcoma (7 cases). Tumors were located in the humerus (5), ulna (1), femur (10) and tibia (17). The mean follow-up was 49.9 months (range 12-127 months). Of the 33 patients, 16 remained disease-free, and 3 were alive with disease. The mean size of the defect after tumor resection was 11.6 cm (range 6-25 cm). Bone union was achieved in 32 patients, with a mean union time of 8.8 months (range 4-18 months). Complications included 1 graft nonunion, 2 infections (1 superficial, 1 deep infection), 1 leg length discrepancy, 2 graft fractures and 3 local recurrences. The mean MSTS score was 87.2% (range 70-100%)., Conclusion: Liquid nitrogen-treated tumor-bearing autograft is an effective option for biological reconstruction after meta-/diaphyseal tumor resection of long bones. This method has excellent clinical outcomes and is especially recommended for patients with no severe osteolytic bone tumors., (© 2022. The Author(s).)

The isomeric enrichment of parahydrogen (pH 2 ) gas is readily accomplished by lowering the gas temperature in the presence of a catalyst. This enrichment is often pursued at two distinct temperatures: ∼51% pH 2 is generated at liquid nitrogen temperatures (77 K), while nearly 100% pH 2 can be produced at 20 K. While the liquid nitrogen cooled generator is attractive due to the low cost of entry, there are benefits to having access to greater than 51% pH 2 for enhanced NMR applications. In this work, we introduce a low-cost modification to an existing laboratory-constructed liquid nitrogen cooled pH 2 generator that provides ∼ 65% pH 2 . This modification takes advantage of vacuum-mediated boiling point suppression of liquid nitrogen, allowing the temperature of the liquid to be lowered from 77 K to nitrogen's triple point of 63 K. The reduced temperature allowed for the generation of parahydrogen fractions of 63-67% at gas flow rates from 20 to 1000 standard cubic centimeters per minute. We compare this to equivalent experiments that did not utilize the temperature-lowering effects of pressure reduction; these controls generally maintained pH 2 fractions of ∼ 50%. All results (experimental and control) agree with the theoretically expected parahydrogen generation at these temperatures. This straightforward modification to an existing pH 2 generator may be of interest to a broad range of scientists involved with parahydrogen research by introducing a simple and low-cost entryway to increased pH 2 fractions using a conventional liquid nitrogen cooled generator., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Brown macroalgae, including the kelp Saccharina latissima, are of both ecological and increasing economic interest. Together with their microbiota, these organisms form a singular entity, the holobiont. Sampling campaigns are required to study the microbiome of algae in natural populations, but freezing samples in liquid nitrogen is complex in the field, particularly at remote locations. Here we tested two simple alternative methods for sampling the microbial diversity associated with the kelp S. latissima: silica gel conservation of tissue and swab samples preserved in DNA/RNA shield solution. We used these techniques to compare apex and meristem samples from Roscoff (Brittany, France) and evaluated their impact on the results of 16S rDNA metabarcoding experiments. Both methods were able to separate apex and meristem microbiomes, and the results were concordant with results obtained for flash-frozen samples. However, differences were observed for several rare genera and ASVs, and the detection of contaminant sequences in the silica gel-preserved samples underline the importance of including blank samples for this method. Globally, our results confirm that the silica gel technique and swabbing combined with DNA/RNA shield preservation are valid alternatives to liquid nitrogen preservation when sampling brown macroalgae in the field. However, they also underline that, regardless of the method, caution should be taken when interpreting data on rare sequences., (Copyright © 2022 Elsevier B.V. All rights reserved.)