Custodiol® Supplemented with Synthetic Human Relaxin Decreases Ischemia-Reperfusion Injury after Porcine Kidney Transplantation.

Objective. Ischemia-reperfusion injury (IRI) is inevitable after kidney transplantation (KT), impairing outcomes. Relaxin-2 (RLX) is a promising insulin-related peptide hormone that protects against renal IRI in rodents, although large animal models are needed before RLX can be tested in a human setting. Methods. In this blinded, randomized, and placebo-controlled experimental study kidneys from 19 donor pigs were retrieved after perfusion with Custodiol® ± RLX (5 or 20 nmol/L) and underwent static cold storage (SCS) for 24 and 48 h, respectively. Subsequently, KT was performed after unilateral right nephrectomy. Study outcomes included markers for kidney function, oxidative stress, lipid peroxidation, and endothelial cell damage. PCR analysis for oxidative stress and apoptosis-related gene panels as well as immunohistochemistry were performed. Results. RLX upregulated SOD2 and NFKB expression to 135% (p = 0.042) and 125% (p = 0.019), respectively, while RIPK1 expression was downregulated to 82% (p = 0.016) of corresponding controls. Further RLX significantly downregulated RIPK1 and MLKL expression and decreased the number of Caspase 3- and MPO-positive cells in grafts after SCS. Conclusions. RLX supplemented Custodiol® significantly decreased IRI via both antioxidant and anti-apoptotic mechanisms. Clinical trials are warranted to implement synthetic human RLX as a novel additive to preservation solutions against IRI.

The Critical Role of Bioenergetics in Donor Cardiac Allograft Preservation.

The traditional philosophy of ex vivo organ preservation has been to limit metabolic activity by storing organs in hypothermic, static conditions. This methodology cannot provide longevity of hearts for more than 4-6 h and is thereby insufficient to expand the number of available organs. Albeit at lower rate, the breakdown of ATP still occurs during hypothermia. Furthermore, cold static preservation does not prevent the permanent damage that occurs upon reperfusion known as ischemia-reperfusion (IR) injury. This damage is caused by increased reactive oxygen species (ROS) production in combination with mitochondrial permeability transition pore (mPTP) opening, highlighting the importance of mitochondria in ischemic storage. There has recently been a major paradigm shift in the field, with emerging research supporting changes in traditional storage approaches. Novel research suggests achieving metabolic homeostasis instead of attempting to limit metabolic activity which reduces IR injury and improves graft preservation. Maintaining high ATP levels and circumventing cold organ storage would be a much more sophisticated standard for organ storage and should be the focus of future research in organ preservation. Given the link between mPTP, Ca2(+), and ROS, managing Ca2(+) influx into the mitochondria during conditioning might be the next critical step towards preventing irreversible IR injury.

Storing Tooth Segments for Optimal Esthetics.

OBJECTIVE: A fractured whole crown segment can be reattached to its remnant; crowns from extracted teeth may be used as pontics in splinting techniques. We aimed to evaluate the effect of different storage solutions on tooth segment optical properties after different durations. STUDY DESIGN: Sixty central incisor crowns were divided into 6 groups (n = 10); Group 1 was kept dry; Groups 2, 3, 4, 5, and 6 were placed in an isotonic solution, water, milk, saliva, and casein-phosphopeptide-amorphous-calcium-phosphate (CPP-ACP), respectively, for 30 min, 12 h, 1 day, 1 week, and 3 weeks. Color values were measured using a colorimeter. Data were analyzed with Kruskal-Wallis tests, Mann-Whitney U-tests, and Friedman Wilcoxon tests with Bonferroni stepwise corrections (p < 0.05). RESULTS: ΔE* values varied from 0.3 to 15.3 over the 3 week period. Group 1 demonstrated the greatest color changes over all durations; Group 6 exhibited the least. L*, a*, b*, and ΔE* values varied between time periods in all groups; the differences were significant (p < 0.01), except for L* and ΔE* values in Group 2 and a* values in Group 6 (p > 0.01). Comparing ΔE* values, Group 6 was significantly different from the other groups for all durations (p < 0.01), except Group 4. CONCLUSIONS: A CPP-ACP complex solution seems a good choice for tooth fragment storage. Milk and saliva solutions may cause perceptible color changes if tooth fragments are stored for 3 weeks before use.

Evaluation of Aloevera Gel as a Storage Medium in Maintaining the Viability of Periodontal Ligament Cells - An in Vitro Study.

OBJECTIVE: To investigate the effectiveness of aloevera gel as a new storage medium in maintaining the viability of periodontal ligament cells. STUDY DESIGN: Premolars extracted for orthodontic reason were obtained. Confluent monolayers of fibroblasts were grown by cell culture method from the PDL cells isolated from the extracted teeth. One ml of this cell suspension was transferred to wells of culture plates, incubated for 24 hrs, followed by exposure to the three experimental media, Hank's balanced salt solution (HBSS), aloevera gel, and packaged drinking water. These plates were then assessed for viable cells using trypan blue dye exclusion test with haemocytometer after 15, 30, 60, 90 and 120 mins. The results obtained were statistically analysed using one-way analysis of variance (ANOVA). RESULTS: At 15 min, HBSS presented maximum mean percentage of viable PDL cells (89%), followed by aloevera at 81% and packaged drinking water at 10%. Aloevera demonstrated 71%, 59%, 57% viable cells at 30, 60, 90 mins respectively. At 120 min, HBSS presented 57% viable cells followed by aloevera gel (45%) and packaged drinking water (3%). No statistical significant difference was observed between HBSS and aloevera gel. CONCLUSIONS: Within the parameters of this study, both aloevera gel and HBSS were effective in maintaining the viability of PDL cells. Hence, aloevera gel could be used as a storage media for avulsed tooth in situations where availability of HBSS is in question.

Custodiol-N, the novel cardioplegic solution reduces ischemia/reperfusion injury after cardiopulmonary bypass.

BACKGROUNDS: On the basis of Custodiol preservation and cardioplegic solution a novel cardioplegic solution was developed to improve the postischemic cardiac and endothelial function. In this study, we investigated whether its reduced cytotoxicity and its ability to reduce reactive oxygen species generation during hypoxic condition have beneficial effects in a clinically relevant canine model of CPB. METHODS: 12 dogs underwent cardiopulmonary bypass with 60 minutes of hypothermic cardiac arrest. Dogs were divided into 2 groups: Custodiol (n = 6) and Custodiol-N (n = 6) (addition of L-arginin, N-α-acetyl-L-histidine and iron-chelators: deferoxamine and LK-614). Left ventricular hemodynamic variables were measured by a combined pressure-volume conductance catheter at baseline and after 60 minutes of reperfusion. Coronary blood flow, myocardial ATP content, plasma nitrate/nitrite and plasma myeloperoxidase levels were also determined. RESULTS: The use of Custodiol-N cardioplegic solution improved coronary blood flow (58 ± 7 ml/min vs. 26 ± 3 ml/min) and effectively prevented cardiac dysfunction after cardiac arrest. In addition, the myocardial ATP content (12,8 ± 1,0 µmol/g dry weight vs. 9,5 ± 1,5 µmol/g dry weight) and plasma nitrite (1,1 ± 0,3 ng/ml vs. 0,5 ± 0,2 ng/ml) were significantly higher after application of the new cardioplegic solution. Furthermore, plasma myeloperoxidase level (3,4 ± 0,4 ng/ml vs. 4,3 ± 2,2 ng/ml) significantly decreased in Custodiol-N group. CONCLUSIONS: The new HTK cardioplegic solution (Custodiol-N) improved myocardial and endothelial function after cardiopulmonary bypass with hypothermic cardiac arrest. The observed protective effects imply that the Custodiol-N could be the next generation cardioplegic solution in the protection against ischemia-reperfusion injury in cardiac surgery.

Effects of a triple antibiotic solution on pulpal dynamics after intentionally delayed tooth replantation in mice.

INTRODUCTION: This study analyzed the detailed biological events underlying pulpal dynamics evoked by 3Mix (the mixture of ciprofloxacin, metronidazole, and minocycline) solution after intentionally delayed tooth replantation because 3Mix improves pulpal healing after tooth injuries. METHODS: The maxillary first molars of 3-week-old mice were extracted and immersed in 3Mix solution for 30 minutes in comparison with phosphate buffered saline (PBS) alone. Cell proliferation, apoptosis, and differentiation were assessed in extracted/replanted teeth during days 0-14 using immunohistochemistry, apoptosis assay, and reverse-transcriptase polymerase chain reaction. RESULTS: 3Mix solution accelerated odontoblast differentiation in the coronal pulp on day 7 and tertiary dentin formation on day 14, whereas the regenerative process was delayed in the PBS group. Cell proliferation and apoptosis occurred in the pulp of the 3Mix group during days 5-7 and subsequently decreased from days 7-14. On day 5, dentin sialophosphoprotein and nestin were first recovered in the 3Mix group, whereas expression levels for alkaline phosphatase, osteopontin, and osteocalcin increased in the PBS group. The expression levels for octamer-binding factor 3/4A and 3/4B reached the maximum level on day 1 and were sharply decreased on day 3 in both groups. High expression levels of Cd11c were first observed in the 3Mix group on day 1 and later at days 5 and 7. CONCLUSIONS: The results suggest that the application of 3Mix may suppress osteoblast differentiation by the migration of dendritic cells to the injury site and via the activation of stem/progenitor cells, resulting in the acceleration of odontoblastlike cell differentiation.

Pedialyte promotes periodontal ligament cell survival and motility.

INTRODUCTION: The search still continues to find the best storage media for avulsed teeth. Unfortunately, some of the recommended storage solutions are not commonly found in households or do not preserve the periodontal ligament (PDL) cells long-term. The purpose of the present study was to determine whether Pedialyte is a viable alternative storage solution for avulsed teeth by assessing its ability to preserve human PDL cell viability. METHODS: Human PDL cells were exposed to 6 different storage solutions (minimal essential medium [MEMα], Hank's balanced salt solution [HBSS], non-fat milk, coconut water, Pedialyte, or tap water) for 2, 6, 24, or 48 hours at 4°C or 25°C. Cell viability was quantified immediately or 1 week after exposure. The effects of these storage solutions on PDL cell motility and bacterial proliferation were also examined. The results were statistically analyzed by analysis of variance. RESULTS: Pedialyte at 4°C and 25°C showed significantly (P < .001) higher cell survival compared with water after all time intervals. No significant difference was noted between control (MEMα), HBSS, coconut water, and Pedialyte at 4°C after 2 hours. Cells stored in Pedialyte for 24 hours at 25°C and assayed 1 week later showed significantly higher cell survivability compared with milk. Pedialyte supported significantly less bacterial growth compared with non-fat milk and coconut water. No difference in cell motility was observed for cells stored for 24 hours in Pedialyte, MEMα, HBSS, milk, or coconut water. CONCLUSIONS: Pedialyte is a viable alternative as a storage solution for avulsed teeth.

Avulsion and storage media.

Avulsion of a tooth caused by trauma, and its successful treatment is a challenging situation that a dentist encounters in clinical practice. There is a definitive treatment for the avulsed tooth, which depends on many factors. The immediate replacement and replantation of the avulsed tooth is necessary, but it cannot always be accomplished for a number of reasons. Thus, the tooth should be transported in a suitable medium to maintain the cell viability. The storage medium is one of the important factors that will help the dentist in rendering successful treatment for the avulsed tooth. There is a continuing search for an ideal storage medium. This review paper focuses on the various storage media, with special reference to coconut water.

Study of the effectiveness of propolis extract as a storage medium for avulsed teeth.

The purpose of the present study was to evaluate the efficacy of propolis extract in maintaining the viability of human periodontal ligament (PDL) cells, and to radiographically analyze tooth replantation and the adjacent periodontium in dogs after storage in this extract. Human PDL cells were incubated with the experimental media propolis, milk, saliva, Hank's balanced salt solution (HBSS), and Dulbecco's modified Eagles medium (DMEM, positive controls), and distilled water (negative control). Cell viability was determined 0, 1, 3, 6, 12, and 24 h later by colorimetric MTT assay. Thirty incisors from dogs were divided into two storage time blocks (1 and 3 h) and were maintained in the experimental media. HBSS served as a positive control, and dry teeth (on gauze) as a negative control. The replanted teeth were radiographed once per month for 6 months. The radiographic images were standardized by the shortening/lengthening factor, and were both qualitatively and quantitatively analyzed. The in vitro results showed that the efficacy of propolis in maintaining functional viability of PDL cells was similar to that of milk. Propolis and milk were significantly better than controls from the 6-h time period. The in vivo results showed that teeth maintained in propolis medium exhibited replacement resorption with significant reduction in tooth length, similar to teeth maintained in saliva and dried teeth. This resorption was less intense with the 3-h storage time than the 1-h storage time. Conditions close to normal were found in teeth maintained in milk, similar to the HBSS control. Therefore, although propolis was effective in maintaining the viability of human PDL cells, resorption of the tooth replantation in dogs occurred under these experimental conditions.

Calcium-supplemented University of Wisconsin solution in long-term myocardial preservation.

The purpose of this study was to assess the effects of the addition of calcium to University of Wisconsin solution in long-term myocardial perfusion. In a heterotopic heart transplantation model, performed in pigs, the donor heart was preserved for 24 hours by means of continuous perfusion in this solution, without (24hUW group) or with calcium, 2.4 mmol/L (24hUW+Ca). During this period, the oxygenation and pH of the solution were measured, as were the calcium and lactate concentrations and enzyme release. After two hours of reperfusion, samples were collected from both ventricles for the morphological study. In the control group, there were no signs that reperfusion had triggered the calcium paradox. The addition of this cation to the preservation solution improved the intercellular junction integrity but, at the same time, favored intracellular calcium overload. This is manifested by increased enzyme release during preservation (LDH: 242+/-95 vs 140+/-25; CK: 668+/-371 vs 299+/-83 (U/L). p<0.01 in both cases) and signs of ventricular contracture: hardness and stiffness were significantly more prominent than in the group without calcium supplementation. Moreover, in comparison with the control group, the structural morphology of 24hUW+Ca is characterized by the more prominent and extensive presence of contraction bands and disorganized actin structure. Thus, under the experimental conditions employed in this study, we consider the addition of calcium to Wisconsin solution to be unadvisable.

[A new transportable machine for the preservation of livers to be transplanted by means of hyperbaric oxygenation perfusion]. / Una nuova macchina trasportabile per la conservazione mediante perfusione iperbarica ossigenata del fegato da trapiantare.

The preservation of livers to be transplanted is currently obtained by static cold storage at 4 C degrees and flushing with UW solution. New methods of preservation are being studied that take advantage of machines for continuous hypothermic perfusion of the organ. Such machines have permitted a lengthening of preservation times and the use of livers from non-beating-heart donors. In an attempt to eliminate the damage due to hypothermia, to lengthen preservation times, and to extend the availability of livers to be transplanted, also using those subjected to short periods of warm ischaemia, we have constructed a transportable machine that produces a hyperbaric atmosphere and allows continuous perfusion of the liver. Ten pig livers from beating-heart donors were perfused with Ringer solution in hyperbaric conditions with oxygen at temperatures ranging from 10 to 25 degrees C for periods of up to 24 hours and studied by means of histopathological analysis and tests of mitochondrial activity (FAU) in order to verify cell viability. The group of livers perfused up to 15 hours yielded an FAU value of 169.40 +/- 5.5 compared to the value of the non-perfused livers (controls) established as 100 and those perfused up to 24 hours had a FAU value of 139.18 +/- 10.7 compared to the controls established as 100, thus demonstrating cell viability. The viability of the organs after preservation with our procedure in the hyperbaric oxygenation perfusion machine gives us good reason to believe that, after appropriate further confirmation of the results, it will be possible to use the machine for the transplantation both of livers subjected to warm ischaemia and of livers preserved for longer periods than is currently the case.

Effect of propolis on survival of periodontal ligament cells: new storage media for avulsed teeth.

Propolis is a multifunctional material used by bees in the construction and maintenance of their hives. Propolis possesses several biologic activities such as anti-inflammatory, antibacterial, antioxidant, antifungal, antiviral, and tissue regenerative, among others. The purpose of this study was to determine the ability of propolis to serve as a temporary storage medium for the maintenance of periodontal ligament (PDL) cell viability of avulsed teeth. PDL cells were obtained from healthy third molars and cultured in Dulbecco's Modified Eagles Medium (DMEM). Cultures were subjected to 10% propolis solution, 20% propolis solution, long-shelf life light milk with lower fat content (milk), Hank's Balanced Salt Solution, tap water as the negative control, and DMEM as the positive control. Tissue culture plates were incubated with experimental media at 37 degrees C for 1, 3, 6, 12, or 24 hours. PDL cell viability was assessed by trypan blue exclusion. Statistical analysis of the data was accomplished by using one-way analysis of variance complemented by the Tukey test. The level of significance was 5% (p<0.05). The results showed that 10% propolis was a more effective storage medium than other groups. In conclusion, propolis can be recommended as a suitable transport medium for avulsed teeth.

The survival of human skin stored by refrigeration at 4 degrees C in McCoy's 5A medium: does oxygenation of the medium improve storage time?

To establish the viable storage time of human skin stored by refrigeration at 4 degrees C in McCoy's 5A medium and to establish whether oxygenating the medium improves the viable storage time, the following experiment was conducted. Eighty discs of human split-thickness skin graft, each 3 mm in diameter, were stored in 40 sterile sealable containers under four different conditions: in 0.9% saline, in McCoy's 5A medium, in oxygenated McCoy's 5A medium, and in carbon dioxide supplemented McCoy's 5A medium. Skin graft viability was assessed using tissue culture. Skin stored in saline was viable for only 1 week, whereas skin stored in McCoy's 5A medium and in oxygenated McCoy's 5A medium was viable for 4 weeks. Skin stored in carbon dioxide supplemented McCoy's 5A solution did not even survive the first week. These findings show that McCoy's 5A medium allows at least 4 weeks of viable human skin storage by refrigeration at 4 degrees C. Furthermore, oxygenating the medium does not seem to improve the viable storage time, and carbon dioxide supplementation is detrimental. The advantages of skin storage by refrigeration and the implications of the above findings are discussed. A clinical case in which split-thickness skin was stored for approximately 5 weeks and still resulted in good graft take is quoted as an example of our experience with the use of McCoy's 5A medium.

Effects of butanedione monoxime and temperature on prolonged cardiac storage.

BACKGROUND: The optimal temperature for cardiac allograft storage remains controversial. We conjectured that supplementation of the potent cardioprotective agent 2,3-butanedione monoxime with calcium may improve allograft storage and make the precise storage temperature less critical. METHODS: Hearts were harvested from Sprague-Dawley rats (250 to 350 g), mounted on a Langendorff apparatus, and instrumented with an intraventricular balloon. Hearts were flushed and stored with either unmodified University of Wisconsin solution (UWS) or UWS supplemented with 10 mmol/L of 2,3-butanedione monoxime and calcium 0.1 mmol/L (BDM). Hearts were then subjected to 12 hours of storage at one of five temperatures (0 degree, 4 degrees, 8 degrees, 12 degrees, or 16 degrees C) in a complete 2 x 5 factorial design (n = 6/group). Data are reported either as a percentage of the prestorage results or as an absolute value (mean +/- standard deviation). RESULTS: Recovery of developed pressure (p < 0.0001), coronary flow (p < 0.0001), and diastolic volume (p < 0.001) were significantly enhanced, whereas creatine kinase (p < 0.0001) and lactate dehydrogenase release (p < 0.0001) were reduced in the BDM versus the UWS groups. In both the BDM and UWS storage groups, recovery was better at temperatures of 8 degrees C or less than at 12 degrees C or more. The single preferred temperature was 4 degrees C, significantly better than 0 degree C with unmodified UWS, while similar to 0 degree and 8 degrees C with BDM. Adenine nucleotide values were decreased equally in the BDM and UWS hearts, but preservation was enhanced at 0 degree C compared with all warmer temperatures. CONCLUSIONS: We conclude that 4 degrees C is the preferred temperature for prolonged cardiac storage with UWS and that the inclusion of 2,3-butanedione monoxime with calcium 0.1 mmol/L markedly enhances recovery for storage temperatures of 8 degrees C or less.