Graft Storage Research Articles

No Association Between Donor Variables and Clinically Significant Outcomes, Reoperations, and Failure After Osteochondral Allograft Transplantation.

Mismatch between osteochondral allograft (OCA) donor and recipient sex has been shown to negatively affect outcomes. This study accounts for additional donor variables and clinically relevant outcomes. To evaluate whether donor sex, age, donor-recipient sex mismatch, and duration of graft storage affect clinical outcomes and failure rates after knee OCA transplantation. Cohort study; Level of evidence, 3. Patients undergoing knee OCA transplantation between 2003 and 2018 were prospectively followed. Inclusion criteria consisted of primary OCA transplantation and minimum 2-year follow-up. Patient descriptive data and allograft donor sex, age, and graft storage time before implantation were collected. Patients were evaluated for reoperation, failure, and achievement of clinically significant outcomes for International Knee Documentation Committee scores. Reoperation was defined as subsequent surgical intervention of the transplanted allograft, including second-look arthroscopy for graft evaluation, debridement, and loose body removal. Failure was defined as revision of the primary OCA transplantation or conversion to arthroplasty. A Kaplan-Meier curve determined cumulative survivability of OCA transplantations, and log-rank testing was used to compare survivorship between groups. Stepwise regression analysis was utilized to evaluate associations between donor variables and achievement of clinically significant outcomes, reoperation, and failure. A total of 372 patients undergoing OCA transplantation were included and followed for a mean 5.4 years (SD, 2.7; range, 2.0-16.3). Isolated OCA transplantation was performed in 45% of cases (169/372). A mismatch in donor and recipient sex was present for more female patients (90%) than male patients (10%; P < .001). Those who had a sex-mismatched graft more frequently underwent concomitant tibial tubercle osteotomy (P = .034). When controlling for patient sex, no other differences were seen between groups matched and mismatched by sex. Univariable and multivariable analysis found no significant difference in survival free from reoperation or failure on the basis of donor-recipient sex mismatch, donor age, or graft storage time before implantation. In contrast to previous historical data, no donor variables were associated with inferior clinical outcomes in patients who underwent OCA transplantation. These data can help inform graft selection, expedient recipient selection, and outcome optimization after OCA transplantation.

24 hours Sub-normothermic Machine Perfusion improves storage of Porcinekidney grafts

24 hours Sub-normothermic Machine Perfusion improves storage of Porcinekidney grafts

Cryomedical technologies as a key to effective decellularization in the creation of scaffolds for vascular transplantation

Background. Cardiovascular diseases (CVDs) rank among the top three causes of death worldwide. In Europe, 3.9 million deaths annually are attributed to CVDs, with 1.8 million occurring among citizens of European Union (EU) countries. The total cost of treating patients with CVDs in EU countries amounts to €210 billion per year. Currently, the primary treatment strategy for patients with advanced stages of CVD remains bypass surgery. A significant increase in demand for vascular grafts over the past decade, particularly small-caliber vessels for cardiovascular bypass procedures, combined with a shortage of donor vessels and the limitations of artificial prostheses, makes the tissue engineering of vascular grafts a high-demand field. Purpose – to characterize modern approaches to creating decellularized vascular scaffolds based on data from open sources. Materials and Methods. The selection of publications was conducted using databases such as PubMed, Clinical Key Elsevier, Cochrane Library, eBook Business Collection, and others, focusing on contemporary methods for creating decellularized scaffolds. The first stage involved searching for literature sources using Keywords: decellularization, extracellular matrix, scaffold. In the second stage, article abstracts were reviewed, and publications not meeting the study criteria were excluded. The third stage involved examining the full texts of selected articles for compliance with inclusion criteria and research relevance. Inclusion criteria for publications subjected to content analysis included: 1) coverage of current information on the creation of decellularized scaffolds; 2) alignment of studies with key principles of evidence-based medicine; 3) open access to the full-text article. Results. The first attempt to create a blood vessel substitute using tissue engineering methods was made by Weinberg C.B. &amp; Bell E. in 1986. Biological scaffolds, composed of extracellular matrix (ECM), are commonly used for various reconstructive surgical procedures and are increasingly employed in regenerative medicine strategies for tissue and organ replacement. ECM is a complex network of macromolecules that provides an appropriate local microenvironment for cell survival and activity in vivo, influencing cell shape, metabolism, function, migration, proliferation, and differentiation. A scaffold, in turn, can be defined as a three-dimensional platform necessary for actions ranging from cell-biomaterial interaction and cell adhesion to controlled biodegradation rates that correspond to tissue regeneration. Decellularization is a method for removing cellular components from organs or tissues to create an acellular scaffold composed of tissue ECM capable of providing a biomimetic microenvironment. The physicochemical signals and biological efficacy of ECM scaffolds can be maintained after decellularization, thus providing a substrate for mechanical support and a biological 3D carrier for subsequent recellularization. The complexity and duration of decellularization protocols are generally proportional to the degree of geometric and biological preservation desired for the tissue after processing. Conclusions. The rising incidence of CVDs and the need for surgical intervention have underscored the necessity, among other things, of creating artificial small-caliber vascular grafts – substitutes for blood vessels, especially those under 6 mm in diameter. Various sources, including human and animal cadavers, have been identified as sources of native vessels for decellularization. The creation of a cellular pattern is a new trend that is actively pursued, directly offering spatial control over angiogenesis, closely mimicking the natural environment. Cryopreservation is one of the most common procedures for graft storage. The freezing-thawing process effectively lyses cells in tissues and organs and is one of the most promising approaches to decellularization in the development of vascular scaffolds.

Unlocking the Promise of Liver Perfusion Technologies for Pediatric Transplantation: A State-of-the-Art Review.

Outcomes after pediatric liver transplantation are generally excellent, but the limited avavailability of suitable, size-matched liver allografts remains a significant barrier. Machine perfusion technology has emerged as a promising approach to expand the donor pool, enabling the use of less ideal whole liver grafts, such as livers donated after circulatory death, and enhancing the execution of split liver transplantation. This review examines the application of machine perfusion in pediatric liver transplantation, focusing on two primary techniques: hypothermic oxygentaed perfusion and normothermic machine perfusion. These methods optimize storage, resuscitation, and assessment of liver grafts before transplantation, potentially expanding the range of usable donor organs. The use of machine perfusion allows for the consideration of suboptimal donor livers and facilitates split liver transplantation, both of which could increase organ availability for pediatric patients. Implementation of machine perfusion could also help reduce waiting list mortality by enabling the safe use of a broader spectrum of donor organs. Adoption of machine perfusion in pediatric liver transplantation will require collaborative, multidisciplinary efforts across transplant centers. By fostering cooperative learning and sharing resources. the integration of machine perfusion into clinical practice has the potential to reduce mortality among children awaiting liver transplantation.

A safe, novel and ingenious method for autogenous bone graft storage in spine surgery with constrained resources - operative site as the bone bank.

The aim of our study is to establish whether the bone graft harvested and stored in the surgical wound by our novel technique is safe, reproducible and preserves the viability of the graft. In doing so, it promises successful bony fusion in spine and orthopaedic surgeries. A prospective clinical case series was conducted for autogenous bone graft storage in complex spine surgeries requiring staged procedures, in resource constrained settings. The bone graft harvested was morselized, wrapped in moist sterile gauze and stored in the paraspinal gutter within the operative site. Thereafter, the surgical wound was easily closed without tension. During the second stage surgery, the stored bone was retrieved and mixed with more autologous/allogenic bone (if necessary) and appropriately laid at fusion sites. Bacterial samples were sent before implantation. 16 complex spinal deformity patients who underwent surgery in a resource constrained hospital over a period of 5 years were included. Duration between both stages was within 2 weeks. All patients showed successful fusion, with mean follow-up of 2.6 years. There were no cases of deep or systemic infection in our series. Surgeons found harvesting, storing and retrieval of graft to be straightforward. The operative site provides an ideal, safe and reproducible location for bone graft storage for staged surgeries conducted in resource constrained situations. The osteogenic potential of the autogenous bone graft is retained. This technique can be extrapolated to other orthopaedic surgeries conducted under resource limited environments like in surgical camps or combat medical facilities.

Descemet Membrane Endothelial Keratoplasty Graft Preparation Using the Liquid Bubble Technique with Subtrabecular Hydrodissection: A Retrospective Real-Life Study

Background: Techniques for the preparation of grafts for Descemet membrane endothelial keratoplasty (DMEK) can be classified into those that involve the manual dissection of the Descemet membrane (DM) and those that use an injection of a liquid or a gas to achieve a separation of the DM from the posterior corneal stroma. The purpose of this study was to evaluate the efficiency of the liquid bubble technique. Methods: The success rate of the technique was calculated retrospectively using the operating reports. Video files for each graft preparation were retrieved and the time, number of injections, and number of injections sites required for the hydrodissection were measured. The number of cases in which a manual dissection of the Descemet membrane was necessary was recorded. Information on donor age and graft preservation time were retrieved from the eye bank file. Results: In 58 cases, the success rate was 98.3%. In the 28 procedures where a video was available, the median time for hydrodissection was 4.4 min. The median number of injection sites was 2, with a median number of injections of 3.5. Manual dissection as a rescue technique was performed in 25% of cases, with one case resulting in graft tears. The mean graft diameter was 7.6 mm. The mean donor age was 66 years, and the mean graft storage time was 22 days. Conclusions: The liquid bubble technique can be a fast and valuable choice for DMEK graft preparation, especially in centers where the tissue is prepared in the operating theater.

Effect of taurine on vascular dysfunction in an in vitro ischemia-reperfusion model of rat thoracic aorta.

The primary objective of this study was to evaluate the protective effect of taurine on endothelial dysfunction in a vascular ischemia-reperfusion (IR) model. Thoracic aortas of 9 male Sprague-Dawley rats (350-500g) were cut into rings and randomized into control (n = 7), IR (n = 8), IR + taurine 1mM (n = 7), IR + taurine 10mM (n = 8), IR + taurine 30mM (n = 8), and IR + taurine 100mM (n = 5) groups. Aortic rings in the IR group were stored in 0.9% saline at 4°C for 24h, placed in Krebs-Henseleit solution gassed with 95%O2 + 5%CO2 at 37°C, and exposed to sodium hypochlorite (200μM) for 30min. Responses to KCl (80mM), phenylephrine (10-10-10-4M), acetylcholine (10-10-10-4M), and sodium nitroprusside (SNP, 10-11-10-5M) were recorded. Emax (maximum response) and pD2 (negative logarithm of concentration producing half-maximum response) were calculated. IR decreased KCl contraction (control 1047 ± 176mg, IR 682 ± 128mg, p = 0.0007), which was reversed by 30 and 100mM taurine (960 ± 313mg, p = 0.02 and 1066 ± 488mg, p = 0.02, respectively). IR impaired phenylephrine, acetylcholine, and SNP responses (p < 0.0001). Taurine did not affect IR-impaired phenylephrine contractions. IR decreased both pD2 (control, 7.1 ± 0.1; IR, 6.0 ± 0.2; p < 0.01) and Emax (control, 83.5 ± 2.7%; IR, 26.8 ± 2.5%; p < 0.0001) of acetylcholine relaxation, both of which were reversed by 100mM taurine (pD2, 7.2 ± 0.1; p < 0.001; Emax, 45.4 ± 2.6%; p < 0.0001). For SNP relaxation, IR decreased pD2 (control 8.2 ± 0.1, IR 7.7 ± 0.1, p < 0.01), which was reversed by 100mM taurine (8.5 ± 0.1, p < 0.0001). Taurine protects endothelial function after IR injury. Further studies should explore the mechanism of this effect and the potential of adding taurine to vascular graft storage solutions.

Ex vivo storage of human osteochondral allografts: Long-term analysis over 300 days using a Ringer-based solution.

Large osteochondral defects are a major challenge in orthopedics, for which osteochondral allograft (OCA) transplantation is nowadays considered as an option, especially in young patients. However, a major issue with OCA is the need for graft storage, which ensures adequate cartilage integrity over time. The aim of this study was to test how long a Ringer-based storage solution can provide good graft quality after explantation and thus meet the requirements for OCA. For this purpose, human osteochondral allografts of the knee and ankle were analyzed. Live/Dead analysis was performed and glycosaminoglycan, as well as hydroxyproline content, were measured as crucial chondrocyte integrity factors. Furthermore, biomechanical tests focusing on stress relaxation and elastic compression modulus were performed. The critical value of 70% living chondrocytes, which corresponds to a number of 300 cells/mm², was reached after an average of 16 weeks of storage. In addition, a constant cell shrinkage was observed over time. The amount of glycosaminoglycan and hydroxyroline showed a slight and constant decrease over time, but no significant differences when compared from Day 0 to the values at Weeks 40-43. Biomechanical testing also revealed no significant differences at the different time points. Therefore, the results show that the Ringer-based storage solution at 4°C is able to provide a chondrocyte survival of 70% until Week 16. This is comparable to previously published storage solutions. Therefore, the study contributes to the establishment of a Ringer-based osteochondral allograft transplantation system for countries where medium-based storage solution cannot be approved.

Time Course of High-Energy Phosphate Depletion During Cold Storage of Human Heart Grafts Using the Celsior Solution.

The aim of this study was to provide insight into high-energy phosphate compound concentration dynamics under realistic clinical cold-storage conditions using the Celsior solution in seven heart grafts discarded from transplantation. The hearts of seven local donors (three males, four females, age 37 ± 17years, height 175 ± 5cm, weight 75 ± 9kg) initially considered for transplantation and eventually discarded were submitted to a Magnetic Resonance Spectroscopy observation in a clinical Magnetic Resonance Imaging scanner over at least 9h. The grafts remained in their sterile container at 4°C during the entire examination. Hence, Phosphocreatine (PCr), adenosine triphosphate (ATP), inorganic phosphate (Pi) and intracellular pH were recorded non-destructively at a 30-minute interval. With the ischemic time Ti, the concentration ratios decreased at PCr/ATP = 1.68-0.0028·Tis, Pi/ATP = 1.38 + 0.0029·Tis, and intracellular pH at 7.43-0.0012·Tis. ATP concentration remained stable for at least 9h and did not decrease as long as phosphocreatine was detectable. Acidosis remained moderate. In addition to the standard parameters assessed at the time of retrieval, Magnetic Resonance Spectroscopy can provide an assesment of the metabolic status of heart grafts before transplantation. These results show how HEPC metabolites deplete during cold storage. Although many parameters determine graft quality during cold storage, the dynamics of HEPC and intracellular pH may be helpful in the development of strategies aiming at extending the ischemic time.

Use of autologous platelet-rich plasma for skin graft preservation: an experimental comparative study.

In this experimental study, we aimed to determine whether platelet-rich plasma (PRP) is a suitable preservative for dermo-epidermal grafts. An additional objective was to investigate how long grafts can be stored without biological degradation. We compared pig skin graft preservation using PRP versus saline solution and crystalloid Custodiol®, which is used for hypothermic preservation of organs for transplantation. Grafts (10 × 10 mm) were placed on gauze impregnated with one of the tested solutions, and stored for 3, 7, 11, and 15 days at a constant temperature of 4°C. We evaluated a total of 240 pig skin samples: 120 by histopathology and 120 by fluorescence optical microscopy. Overall, Custodiol® solution appeared to be the best medium for preservation of dermo-epidermal grafts, with beneficial properties manifested on days 7 and 11. Although we expected PRP to be a better preservative than saline, this was not confirmed by our results, as we found no significant difference between these two media. In fact, by day 3, the histopathological results were better with standard saline solution than with PRP. On day 15, with each tested solution, some samples showed histological changes that are incompatible with graft viability. Overall, Custodiol® appears to be the best medium for dermo-epidermal graft preservation. Moreover, the present findings suggest a maximum graft storage time of 11 days in all of the tested solutions. We do not recommend using grafts stored for 15 days, due to isolated signs of graft biodegradation with all solutions.

Loss of Autophagy Activity during Storage of Hematopoietic Stem Cell Grafts Is Associated with Reduced Potency

Maximizing the potency of hematopoietic stem cell (HSC) grafts including cord blood (CB) units (CBU) is essential to minimize risk of engraftment delays and failures. Low cell dose in CB transplantation is partially responsible for the slower engraftment but processing delays could also be at play since CBU can be stored at room temperature (RT) up to 48 hours before cryopreservation. We hypothesized that such prolong storage reduces graft potencies due to a loss in hematopoietic stem cell and progenitors (HSPC) content. CBU were used as HSC graft. Units were split in 2 halves; one processed shortly after collection (&amp;lt;15 hrs, fresh) or after storage at RT (~43 hrs, stored). The impact of storage on graft quality was assessed using standard assays and bioinformatic analysis used to identify molecular pathways impacted by storage. Monitoring assays revealed that all CBU samples passed the minimal FACT-criteria threshold for post-thaw CD34+ cell viability and potency. However, the net number of ISHAGE CD34+ cell counts were reduced in stored samples (-20%, p&amp;lt;0.01). The colony forming-unit (CFU) assay used to measure the impact of storage on potency also revealed a 20% reduction in CFU in stored samples (&amp;lt;0.05, n=7). Losses in potency was confirmed with a second assay, the IL-3-phospho-STAT5 (pSTAT5) assay, which revealed a 10-fold loss in STAT5 signalling in stored vs. fresh CD34+ cells (p&amp;lt;0.0001, n=3). We hypothesized that factors released by CB could be responsible for the reduction of HSPC. In support of this, plasma isolated from stored CBU induced greater apoptosis of CD34+ cells than fresh plasma (+30% AnnexinV+ cells, p&amp;lt;0.01, n=3). This translated into losses of potency detectable after 30 minutes (CFU assay, p&amp;lt;0.001) and with further reduction over time peaking around 2 hours. Next, we sought to identify the molecular programs that govern this detrimental effect within CD34+ cells. Towards this we performed RNAseq analysis of CD34+ cells exposed to fresh or stored plasma for 20 minutes and 4 hours. A time course comparative analysis of differentially expressed genes (DEG, Log 2Fold &amp;gt;1.5, and q&amp;lt;0.05) between cells exposed to fresh vs stored plasma identified a total of 810 DEG genes. Gene ontology enrichment analysis identified autophagy, cell cycle, histone and DNA methylation and mRNA regulation as the major molecular programs that might be up regulated by the paracrine factors from fresh plasma but repressed by those from stored plasma. Autophagy flux assay confirmed that CD34+ cells isolated from stored UCB samples had reduced autophagy activity, and that stored plasma induced a 50% reduction in autophagy flux (p&amp;lt;0.001, n=3). Furthermore, RT-qPCR analysis confirmed that prolonged storage of CBU samples at RT increased the cellular senescence marker p21(CDKN1B), down regulated cell cycle genes ( CDK4 and CDK7) and autophagy genes ( ATG4, ATG12 and BECN1). We hypothesized that re-activation or prevention of autophagy loss could prevent losses of HSPC within CBUs stored at RT. To test this, CBUs were divided and individually supplemented with either autophagy activators (rapamycin or trehalose), autophagy inhibitor (3-methyladenine, 3-MA) or DMSO control and stored up to 43 hours. As expected, the early autophagy inhibitor 3-MA failed to restore HSPC numbers and potency. However, addition of trehalose prior to storage restored 99% of both net number and potency of HSPCs in HSC grafts as measured by the ISHAGE CD34+ counts, CFU and pSTAT5 assays to baseline level (n=3). Interestingly, the near complete restoration mediated by trehalose coincided with restauration of autophagy activity, repression of senescence gene CDKN1B and activation of cell cycle genes. In contrast, rapamycin only partially restored HSPC numbers and potency which coincided with the activation of autophagy genes but not others mentioned above. In conclusion, the loss of potency and viability seen in CBU grafts originates in part by paracrine-mediated mechanisms that lead to loss of autophagy, down regulation of cell cycle regulators and induction of senescence in HSPCs. Interestingly, addition of trehalose as a natural supplement precludes these molecular changes and restores CBU potency during storage. Taken together, these results stress the importance of rapid processing of HSC grafts and identify an attractive new solution to maintain high HSC graft potency post-collection during storage at ambient temperature.

Correction to: 78 Improvement of Cell Viability in Temporary Storage of Split Thickness Skin Grafts.

Correction to: 78 Improvement of Cell Viability in Temporary Storage of Split Thickness Skin Grafts.

The Effect of Various Temperatures on the Inflammatory Profile of Fat Graft Storage: An Experimental Study.

Fat tissue has been widely used as a filler material during plastic surgery, but unpredictable fat retention remains a significant concern. Fat tissue is vulnerable to ischemia and hypoxia, but it always has waiting time before injection in the operation theater. Apart from transferring fat tissue as quickly as possible after harvesting, washing the aspirate with cool normal saline is often used. However, the mechanisms of cool temperature acting on adipose tissue have yet to be fully elucidated. Herein, this study aims to explore the effect of preservation at different temperatures on the inflammatory profile of adipose tissue. Inguinal adipose tissue of rats was collected and cultured in vitro under 4°C, 10°C, and room temperature for 2 hours. The proportion of damaged adipocytes and an array of cytokines were determined. We observed that the damage rate of the adipocyte membrane was slightly higher at room temperature, but there was no significant difference, while we noticed increased IL-6 and MCP-1 levels in adipose tissue at room temperature ( P ＜0.01). The 4°C and 10°C cool temperatures may offer protection against proinflammatory states during the adipose tissue preserved in vitro.

CARD27: Morphologic and functional assessment of beating heart in normothermic ex-situ preservation

Background: Heart transplantation remains the treatment of choice for patients experiencing end-stage heart failure. The emergence of cardiac ex situ machine perfusion not only facilitates the use of donor heart through the avoidance of potentially damaging ischemia during graft storage, it also opens new horizons for more sensitive monitoring of graft quality in the transplant field, and morphologic-functional assessment. Methods: Twenty-five domestic adult pigs, weighing 93.1±13.1kg were selected for this study. Until cardiectomy, all pigs received Principles of Laboratory Animal Care. The pig heart were cannulated, and then connected to the Portable System for Normothermic Ex-Situ HeartPreservation (our made). In the Heart Preservation System (HPS), oxygenated blood is perfused by a centrifugal pump into the aorta, perfusing the coronary arteries. After the end of experiment, (6-14 hour) pig heart was arrested with normothermic blood cardioplegia and disconnected. The pig isolated heart was conditioned with Levosimendan while in the system and hemofiltration was applied in the HPS in order to protect and improve donor heart function. Ex-Situ Heart Perfusion process was accompanied with morphologic-functional assessment such as echocardiography, contrast-enhanced magnetic resonance, thermodilution technique and biochemistry. Results: Mean (SD) ischemic time was 19.2 (±3.3) min. Mean ex vivo perfusion time was 560 (±143) min. Time of sinus rhythm restoration was 2.3 (5.7) min. All isolated hearts had stable perfusion, biochemical, as well as myocardial functional parameters measured by echocardiography and thermodilution technique, and histological characteristics in the perfusion system. MRI structural images showed no infiltrates alterations in myocardium. Mean venous lactate trend are with normal levels at the end of perfusion 1.6 (±0.6). Conclusion: Normothermic Ex-Situ Heart Perfusion provides near to physiologic, reproducible approach to sustain the isolated heart for long period of time. Multiparametric assessment of organ morphology and function is feasible in Ex-Situ Heart Perfusion, and may improve organ assessment, leading to a better selection for transplant.Figure 1. Simplified schema of the experimental design and protocol.

PULM8: Morphologic And Functional Assessment Of Breathing Lungs In Normothermic Ex-Situ Preservation

Background: Continuous ex-situ machine perfusion has been proposed as an alternative and superior method of donor lungs preservation compared to cold static storage especially during the transport time. The emergence of cardiac ex situ machine perfusion not only facilitates the use of donor lung through the avoidance of potentially damaging ischemia during graft storage, it also opens new horizons for more sensitive monitoring of graft quality in the transplant field, and morphologic-functional assessments. Methods: Twenty-five domestic adult pigs, weighing 93.1±13.1kg were selected for this study. Until pulmonectomy, all pigs received Principles of Laboratory Animal Care. The pig lung were cannulated, and then connected to the Portable System for Normothermic Ex-Situ Lung Preservation (our made). In the Lung Preservation System (HPS), oxygenated blood is perfused by a centrifugal pump into the pulmonary artery and ventilation occurs. After the end of experiment, (6-14 hour) pig lung is disconnected. The pig isolated lung was connected to hemofiltration while in the system and applied in the HPS in order to protect and improve donor lung function. Ex-Situ Lung Perfusion process was accompanied with morphologic-functional assessments such as bronchoscopy, computerized tomography. Results: Mean (SD) ischemic time was 26.2 (±5.3) min. Mean ex vivo perfusion time was 560 (±110) min. All isolated lungs had normal ventilation parameters. All isolated lung had stable perfusion, biochemical and histological characteristics in the perfusion system. Conclusion: Normothermic Ex-Situ Lungs Perfusion provides more physiologic and reproducible approach to sustain the isolated organs.

78 Improvement of Cell Viability in Temporary Storage of Split Thickness Skin Grafts

Abstract Introduction Autologous split thickness skin grafts (STSGs) are the preferred donor tissue for restoration of large tissue deficits. Although grafting should be done at the time of donor harvest, various factors necessitate tissue storage, traditionally in saline solution (SS) at 4oC. However, STSGs often expire before grafting in these conditions. Patients then require further excision procedures or die due to inadequate skin coverage. For patients’ wellbeing, establishing a new storage protocol is imperative. This study sought to determine an effective storage media for prolongation of graft viability for subsequent incorporation into excision and grafting practice. Methods Following patient consent, excess tissue &amp;gt;1cm2 was collected after grafting procedures. Tissue was stored at 4oC in RPMI, DMEM, and SS with penicillin/streptomycin. Samples were evaluated at 2, 5, 7, and 14 days. Cell viability was measured by Trypan blue dye exclusion test in an automated cell counter yielding percentage of viable keratinocytes (PVK). An unbiased pathologist assessed tissue integrity by epidermal-dermal junction, perinuclear keratinocyte halos, and collagen organization with higher scores denoting deterioration. Data were evaluated by ANOVA and Effect size. Results A total of 155 sets of two specimens each were collected from 13 patients. Mean PVK values at day 2, 5, 7, and 14: SS (57.38, 36.96, 38.38, 27.50), RPMI (69.46, 69.92, 70.33, 62.67), DMEM (63.17, 53.50, 54.83, 37.92). By pairwise comparison, RPMI preserved PVK better than SS at days 5, 7, and 14 (p = 0.001 for each day). DMEM preserved PVK better than SS when comparing all days (p = 0.011) but failed to produce a statistically significant difference at any specific day. RPMI preserved PVK greater than DMEM each day. However, statistically significant differences were only revealed at day 14 (p = 0.017) and when comparing all days (p &amp;lt; 0.001). Effect size was large for overall group differences of PVK at day 5, 7, and 14 (Cohen’s f = 0.72, 0.73, and 0.75). Tissue integrity scores at day 2, 5, 7, and 14: SS (0.69, 1.0, 1.38, 2.0), RPMI (0.54, 0.92, 0.85, 1.54), DMEM (0.77, 0.92, 0.77, 1.15). Although culture media preserved tissue integrity greater than SS, no statistically significant differences were found. Conclusions RPMI preserved cell viability better than DMEM and SS. This suggests that STSGs stored in RPMI may prolong the time of which tissue could be stored and successfully amalgamated with skin. Applicability of Research to Practice Storing tissue in RPMI could benefit burn patients by decreasing tissue disposal therefore reducing future donor site area or eliminating repetitious excision procedures entirely. These effects decrease inpatient days and healthcare costs. Most importantly, successful tissue storage will save patients’ lives, especially those of large surface area burns who lack sufficient donor sites.

Osteochondral allograft transplantation of the knee: a review of indications, techniques, outcome and how to promote biology

Articular cartilage lesions of the knee in young and active patients can result in significant disability. Osteochondral allografts (OCAs) can be used to treat challenging articular lesions of the knee ≥2 cm2, either as a primary or revision procedure. They have the advantage of restoring the articular surface with mature hyaline cartilage without any size limitations or donor site morbidity. Chondrocyte viability above 70% at the time of implantation is thought to be desirable, and consequently OCA implantation within the 28-day graft expiration date can result in logistical challenges. No randomized trials exist; however, excellent clinical outcomes have been reported, along with good long-term survivorship up to 20 years. Several factors can influence the outcome after OCA use, including patient selection, anatomical location, associated pathologies and several biological considerations. The purpose of this article is to review the latest evidence on treatment indications, graft storage and surgical techniques, biological considerations, patient outcomes and rehabilitation after surgery.

Analysis of Temperature During Storage and the Period of Warm Ischemia of the Graft in Kidney Transplants

Objective: This study aims to analyze the temperatures of kidney transplants performed at Hospital Municipal São José, Joinville/SC, throughout the period of cold ischemia and whether this temperature is in line with what is recommended in the literature. Method: Prospective analysis based on all kidney grafts that was used for kidney transplant in the unit of kidney transplant of Hospital Municipal São José. Results: The study analyzed that the current method of graft conservation partially meets the expected values, to maintain the recommended temperature. Conclusion: It is important to maintain studies and analyzes on the subject for continuous improvement in graft storage.

Methane Admixture Protects Liver Mitochondria and Improves Graft Function after Static Cold Storage and Reperfusion

Mitochondria are targets of cold ischemia-reperfusion (IR), the major cause of cell damage during static cold preservation of liver allografts. The bioactivity of methane (CH4) has recently been recognized in various hypoxic and IR conditions as having influence on many aspects of mitochondrial biology. We therefore hypothesized that cold storage of liver grafts in CH4-enriched preservation solution can provide an increased defence against organ dysfunction in a preclinical rat model of liver transplantation. Livers were preserved for 24 h in cold histidine-tryptophan-ketoglutarate (HTK) or CH4-enriched HTK solution (HTK-CH4) (n = 24 each); then, viability parameters were monitored for 60 min during normothermic isolated reperfusion and perfusate and liver tissue were collected. The oxidative phosphorylation capacity and extramitochondrial Ca2+ movement were measured by high resolution respirometry. Oxygen and glucose consumption increased significantly while hepatocellular damage was decreased in the HTK-CH4 grafts compared to the HTK group. Mitochondrial oxidative phosphorylation capacity was more preserved (128.8 ± 31.5 pmol/s/mL vs 201.3 ± 54.8 pmol/s/mL) and a significantly higher Ca2+ flux was detected in HTK-CH4 storage (2.9 ± 0.1 mV/s) compared to HTK (2.3 ± 0.09 mV/s). These results demonstrate the direct effect of CH4 on hepatic mitochondrial function and extramitochondrial Ca2+ fluxes, which may have contributed to improved graft functions and a preserved histomorphology after cold IR.

Effect of Copper on the Function of Isolated Porcine Kidneys Stored Using Simple Hypothermia.

Renal ischemia in the peri-transplant period causes a number of changes that adversely affect the initiation of normal vital functions in grafts after transplantation. To minimise the extent of ischemic damage, organs are stored in preservation fluid. The components of the fluid are supposed to ensure stabilisation of the cell cytoskeleton, protect against oxygen free radicals, reduce cell swelling, and ensure endothelial cell integrity. The aim of this study was to analyse the protective effect of Cu2+, as a component of Biolasol preservative fluid, in the prevention of nephron damage occurring during the graft storage period. Analyses of the effectiveness of copper in the presence of prolactin added to Biolasol fluid were also carried out. Forty isolated pig slaughter kidneys were used in the study, avoiding the use of laboratory animals. The kidneys were stored using simple hypothermia. After 2 h and 48 h of graft storage, selected biochemical indicators of renal function were determined in the collected perfusates. The addition of Cu2+ at a dose of 1 µg/L to the composition of Biolasol fluid was found to affect the generation of ischemic damage in the isolated pig kidney. The intensity of the occurrence of these processes is exacerbated by the presence of prolactin at a dose of 0.1 µg/L.