Development and validation of cryopreserved or freeze-dried decellularized human dermis for transplantation.

For decades, dermal tissue grafts have been used in various regenerative, reconstructive, and augmentative procedures across the body. To eliminate antigenicity and immunogenic response while still preserving the individual components and collective structural integrity of the extracellular matrix (ECM), dermis can be decellularized. Acellular dermal matrix (ADM) products like such are produced to accurately serve diverse clinical purposes. The aim of the present study is to evaluate the efficacy of a novel decellularization protocol of the human dermis, which eliminates residual human genetic material without compromising the biomechanical integrity and collagenous content of the tissue. Moreover, a freeze-drying protocol was validated. The results showed that though our decellularization protocol, human dermis can be decellularized obtaining a biocompatible matrix. The procedure is completely realized in GMP aseptic condition, avoiding tissue terminal sterilization.

Decellularized cryopreserved human pericardium: a validation study towards tissue bank practice.

Pericardial patches are currently used as reconstructive material in cardiac surgery for surgical treatment of cardiac septal defects. Autologous pericardial patches, either treated with glutaraldehyde or not, can be used as an alternative to synthetic materials or xenograft in congenital septal defects repair. The availability of an allogenic decellularized pericardium could reduce complication during and after surgery and could be a valid alternative. Decellularization of allogenic tissues aims at reducing the immunogenic reaction that might trigger inflammation and tissue calcification over time. The ideal graft for congenital heart disease repair should be biocompatible, mechanically resistant, non-immunogenic, and should have the ability to growth with the patients. The aim of the present study is the evaluation of the efficacy of a new decellularization protocol of homologous pericardium, even after cryopreservation. The technique has proven to be suitable as a tissue bank procedure and highly successful in the removal of cells and nucleic acids content, but also in the preservation of collagen and biomechanical properties of the human pericardium.

Validation of orthopedic allograft preparation process: a new application of media fill test.

Musculoskeletal allografts represent an important practice in orthopedic surgeries and the demand for them has been growing. For this reason, in order to reduce clinical risk and to more efficiently manage the increase of allograft usage and also to optimize timing of the surgeries, the thawing and washing processes with aseptic technique were centralized in the department of Hospital Pharmacy. This study describe the design and execution of an adapted Media Fill Test (MFT) to demonstrate aseptic thawing and washing of allografts. For this specific and innovative setting, to better simulate the actual processing steps, a surrogate system was developed to simulate the tendon allograft. The aseptic technique of four operators was assessed and an initial performance validation and the first revalidation were described. All MFT were completed successfully, with no observation of turbidity. The readapted MFT shown in this study can provide insight into this innovative and growing field to other health professionals who want to implement this service.

Characterization of Human Subcutaneous Adipose Tissue and Validation of the Banking Procedure for Autologous Transplantation.

Adipose tissue (AT) is composed of a heterogeneous population which comprises both progenitor and differentiated cells. This heterogeneity allows a variety of roles for the AT, including regenerative functions. In fact, autologous AT is commonly used to repair soft tissue defects, and its cryopreservation could be a useful strategy to reduce the patient discomfort caused by multiple harvesting procedures. Our work aimed to characterize the cryopreserved AT and to validate its storage for up to three years for clinical applications. AT components (stromal vascular fraction-SVF and mature adipocytes) were isolated in fresh and cryopreserved samples using enzymatic digestion, and cell viability was assessed by immunofluorescence (IF) staining. Live, apoptotic and necrotic cells were quantified using cytometry by evaluating phosphatidylserine binding to fluorescent-labeled Annexin V. A multiparametric cytometry was also used to measure adipogenic (CD34+CD90+CD31-CD45-) and endothelial (CD34+CD31+CD45-) precursors and endothelial mature cells (CD34-CD31+CD45-). The maintenance of adipogenic abilities was evaluated using in vitro differentiation of SVF cultures and fluorescent lipid staining. We demonstrated that AT that is cryopreserved for up to three years maintains its differentiation potential and cellular composition. Given our results, a clinical study was started, and two patients had successful transplants without any complications using autologous cryopreserved AT.

Stapled fascial suture: ex vivo modeling and clinical implications.

BACKGROUND: Recently, in the field of abdominal wall repair surgery, some minimally invasive procedures introduced the use of staplers to provide a retromuscular prosthetic repair. However, to the knowledge of the authors, there are little data in the literature about the outcomes of stapled sutures adoption for midline reconstruction. This study aims to investigate the biomechanics of stapled sutures, simple (stapled), or oversewn (hybrid), in comparison with handsewn suture. From the results obtained, we tried to draw indications for their use in a clinical context. METHODS: Human cadaver fascia lata specimens, sutured (handsewn, stapled, or hybrid) or not, underwent tensile tests. The data on strength (maximal stress), ultimate strain (deformability), Young's modulus (rigidity), and dissipated specific energy (ability to absorb mechanical energy up to the breaking point) were recorded for each type of specimens and analyzed. RESULTS: Stapled and hybrid suture showed a significantly higher strength (handsewn 0.83 MPa, stapled 2.10 MPa, hybrid 2.68 MPa) and a trend toward a lower ultimate strain as compared to manual sutures (handsewn 344%, stapled 249%, hybrid 280%). Stapled and hybrid sutures had fourfold higher Young's modulus as compared to handsewn sutures (handsewn 1.779 MPa, stapled 7.374 MPa, hybrid 6.964 MPa). Handsewn and hybrid sutures showed significantly higher dissipated specific energy (handsewn 0.99 mJ-mm3, stapled 0.73 mJ-mm3, hybrid 1.35 mJ-mm3). CONCLUSION: Stapled sutures can resist high loads, but are less deformable and rigid than handsewn suture. This suggests a safer employment in case of small defects or diastasis (< W1 in accord to EHS classification), where the presumed tissutal displacement is minimal. Oversewing a stapled suture improves its efficiency, becoming crucial in case of larger defects (> W1 in accord to EHS classification) where the expected tissutal displacement is maximal. Hybrid sutures seem to be a good compromise.

Human amniotic membrane: an improvement in the treatment of Medication-related osteonecrosis of the jaw (MRONJ)? A case-control study.

The aim of this article is to report the results obtained by the use of HAM in surgical wound healing and the reduction of relapse in patients affected by Medication-related osteonecrosis of the jaw (MRONJ).The study involved patients with the diagnosis of MRONJ, surgically treated between October 2016 and April 2019, in a case-control setting. Enrolled patients were randomly divided into 2 groups. One group will be treated with resective surgery and with the insertion of HAM patch (Group A), while the second group had been treated exclusively with resective surgery (Group B).The patients underwent MRONJ surgical treatment with the placement of amniotic membrane patches at the wound site. Data regarding the long-term complications/functions were evaluated at 3, 6, 12, and 24 months after surgery. Pain measurements were performed before the intervention (T0), 7(T1) and 30(T2) days after surgery. 49 patients were included in the study. 2 patients of GROUP A after 30 days since they were surgically treated showed persistent bone exposure. 5 patients of group B demonstrated a lack of healing of the surgical wound with the persistence of bone exposed to 30 days after surgery. Statistical analysis ruled out any difference in OUTCOME (relapse) between GROUP A and B (p = 0.23). However, the Fisher test highlighted a significant difference between the use of HAM and only surgical treatment in pain at rest (p = 0.032). The use of amniotic membrane implement the patient's quality of life and reduce pain perception. has a learning curve that is fast enough to justify its routine use.

Computer-assisted surgery with custom prostheses and human amniotic membrane in a patient with bilateral class IV TMJ reankylosis: a case report.

A gold-standard technique has yet to be found for the treatment of temporomandibular joint ankylosis (TMJa), particularly in patients with recurring ankylosis. A 58-year-old male patient, with a history of multiple TMJ surgeries and severe limitation of mouth opening (maximum interincisal distance [MID] was 10 mm). Computerised tomography (CT) imaging highlighted a bilateral type IV ankylosis. The surgical guides were manufactured using a 3D printing method after obtaining a proper design of the osteotomy lines. The positioning of the fossa and condyle components of the custom TMJ prosthesis was digitally performed. Osteotomies were carried out using surgical guides and TMJ prostheses were placed as per the virtual planning. A human amniotic mambrana is inserted between the two prosthetic components to avoid ranchylosis. The post-operative CT showed the correct positioning of the condylar prosthesis. MID after 10 days was 37 mm. Total joint reconstruction surgery using 3D virtual surgical planning may be an effective surgical option for achieving a precise surgical outcome and making use of a single-stage approach in cases of TMJa and the use of the amniotic membrane, thanks to its healing properties and reduction of pain perception, seems to improve the quality of the immediate post-operative period.

Cryopreservation of human amniotic membrane for ocular surface reconstruction: a comparison between protocols.

PURPOSE: To compare the effects on adhesive and structural properties of newer preservation conditions to those obtained with an established, standardized protocol (dimethyl sulfoxide at -180 °C). In attempt to simplify and enhance the safety of the procedure, we tested dextran-based freezing medium and a dry condition (no medium) at temperatures of -80 °C. METHODS: Five patches of human amniotic membrane were obtained from three different donors. For each donor, five preservation condition were tested: dimethyl sulfoxide at -180 °C, dimethyl sulfoxide at -80 °C, dextran-based medium at -180 °C, dextran-based medium at -80 °C and dry freezing at -80 °C (no medium). At the end of four months storage period, adhesive properties and structure were analyzed. RESULTS: None of the newer preservation protocols showed differences in adhesive and structural properties of the tissues. The stromal layer always kept its adhesiveness, while both structure and basement membrane were not altered by any the preservation protocol. CONCLUSIONS: Switching from liquid nitrogen cryopreservation to -80 °C would reduce manipulation, simplify the procedure, making it also cheaper. The use of dextran-based freezing medium or no medium at all (dry condition) would avoid the potential toxicity of the dimethyl sulfoxide-based freezing media.

Impact of Sars-CoV-2 pandemic on the Veneto Region multitissue bank activity.

Covid pandemic affected donation activities worldwide, especially for living donation due to the lack of elective surgery. Moreover, the number of heart-beating and non-heart beating donors has recorded a decrease. Fondazione Banca dei Tessuti di Treviso (FBTV) is a non-profit healthcare organisation, located in Veneto Region, tasked with procurement, processing, preserving, validating and distributing human tissue for clinical use. During Covid-19 outbreak, operations in FBTV have never stopped and a great effort was required to maintain a standard trend of activity. The aim of this study was to describe the impact of Sars-CoV-2 on the activity of a multitissue bank in Italy. Moreover, we investigated the presence of the virus in tissues retrieved from two Sars-CoV-2 positive cadaver donors. Our survey demonstrated that the transplantation network of Veneto Region has positively reacted to the pandemic scenario, thanks to the effort of all personnel involved. Statistical analyses underlined that most of the activities of the tissue bank were unaffected during the Sars-CoV-2 pandemic.

Bench thrombolysis and "autotransplantation" as a rescue treatment for venous thrombosis after living-donor kidney transplantation.

BACKGROUND: Allograft venous thrombosis is a severe complication after kidney transplantation (KT). Early diagnosis and prompt treatment are crucial in preserving the survival of the allograft. In this study, we aimed to describe an emergent strategy for the management of acute allograft venous thrombosis. CASE PRESENTATION: A 4-year-old girl, weighing 13.5 kg, was diagnosed with bilateral congenital renal hypodysplasia, urogenital sinus and anorectal malformation. The patient was referred to our department for living-donor KT. Her mother was eligible as a donor, presenting a body weight ratio of 1:4.5. Thrombosis of the inferior vena cava (ICV) was also identified, without any predisposing factor for thrombophilia. KT was performed by an extraperitoneal approach without complications. Venous anastomosis required a human vascular graft sutured to the ICV, and renal artery was anastomosed to the aorta. On postoperative day (POD) 8, acute abdominal pain and hematuria led to the diagnosis of an allograft venous thrombosis. An emergent laparotomy was required to explant the allograft, followed by bench surgery. The allograft was irrigated with thrombolytic agents and lactated Ringer's solution and then after removing the venous vascular graft, it was reimplanted through vascular anastomosis with the ICV and aorta. The recovery of perfusion and function was good with diuresis since day 4 after re-surgery. At 2-year follow-up, the child presented normal allograft function with an estimated GFR of 65 ml/min/1.73 m2 . CONCLUSION: According to our experience, explantation of the kidney allograft, followed by irrigation with thrombolytics in bench surgery, and reimplantation resulted in unexpected optimal outcomes in the case of allograft venous thrombosis.

Contamination profile in allografts retrieved from multitissue donors: longitudinal analysis.

Microbiological contamination of retrieved tissues has become an issue of key importance and is a critical aspect of allograft safety, especially in the case of multi-tissue donations, which frequently become contaminated during retrieval and handling. We analysed contamination in 11,129 tissues with a longitudinal contamination profile for each individual tissue. Specifically, 10,035 musculoskeletal tissues and 1094 cardiovascular tissues were retrieved from a total of 763 multi-tissue donors, of whom 105 heart-beating organ donors and 658 deceased tissue donors. Of the 1955 tissues found to be contaminated after the first decontamination step, 1401 tissues (72%) were contaminated by the same species as the one(s) isolated at retrieval (Time1) and 554 (28%) by different species. Among the 113 tissues testing positive after the 2nd decontamination (Time3), 36 tissues (32%) were contaminated by the same species detected at Timel while the contaminating species differed from Time1 in 77 tissues (68%). The higher the number of contaminating species per tissue the higher the percentage of tissues in which contamination changed over time compared to Time1. The analysis revealed a 28% incidence of new species in tissues already testing positive after retrieval and of 3.5% of tissues becoming positive after admission to the tissue bank. Of these, coagulase-negative Staphylococcus accounted for over 70% of new contaminations.

Stability analysis of the antibiotic cocktail used by Treviso Tissue Bank Foundation for tissues decontamination.

Although careful donor selection reduces tissue contamination, close microbiological control of harvested allografts remains a key task of tissue banks. To guarantee the safety of human tissues for allograft transplantation, a decontamination regimen must be adopted which, as recommended by European guidelines, is active against the majority of microorganisms isolated in tissues. Antibiotic decontamination methods differ from one tissue bank to another in terms of antimicrobial agents, temperature and length of exposure. After identifying the most effective antibiotics against the bacterial strains most commonly isolated in allografts, Treviso Tissue Bank Foundation demonstrated the efficacy of an antibiotic cocktail for tissue decontamination containing Gentamicin, Vancomycin and Meropenem. The aim of this study was to analyse the degradation kinetics of the three antibiotics according to preparation method and use. The results show that only Meropenem is unstable at + 4 °C, while Gentamicin and Vancomycin are valid for over 10 days. We thus established to add Meropenem before the start of the tissue decontamination phase.

Cytokine expression and ultrastructural alterations in fresh-frozen, freeze-dried and γ-irradiated human amniotic membranes.

The aim of this work was to compare the effects on human amniotic membrane of freeze-drying and γ-irradiation at doses of 10, 20 and 30 kGy, with freezing. For this purpose, nine cytokines (interleukin 10, platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, epidermal growth factor, transforming growth factor beta 1, and tissue inhibitors of metalloproteinase-1, -2, and -4) were titrated in 5 different preparations for each of 3 amniotic membranes included in the study. In addition, the extracellular matrix structure of each sample was assessed by transmission electron microscopy. While freeze-drying did not seem to affect the biological structure or cytokine content of the different amniotic membrane samples, γ-irradiation led to a significant decrease in the tissue inhibitors of metalloproteinase-4, basic fibroblast growth factor and epidermal growth factor, and induced structural damage to the epithelium, basement membrane and lamina densa. The higher the irradiation dose the more severe the damage to the amniotic membrane structure. In conclusion, the Authors recommend processing amniotic membrane under sterile conditions to guarantee safety at every step rather than final sterilization with γ-irradiation, thereby avoiding alteration to the biological characteristics of the amniotic membrane.

Amniotic membranes in ophthalmology: long term data on transplantation outcomes.

The use of amniotic membrane (AM) is a widespread clinical practice for eye surgeries and the treatment of an increasing number of ocular surface pathologies. Here we describe the AM collection methods and donor selection criteria adopted by our tissue bank to distribute 5349 amniotic membrane patches over the last 12 years for the treatment of several ocular pathologies. Specific quality control measures are described and the long term results attained using the reported procedure are presented. A case of AM utilized to treat severe ocular ulceration is also described as an example of AM transplantation. Collective data for the total amniotic membrane patches deployed to treat various ocular diseases are discussed and success rates for AM transplantations are reported. An extensive follow-up is illustrated. The results suggest that the procedures and protocols used by the Treviso Tissue Bank Foundation and Veneto Eye Bank Foundation for collection, preservation, distribution and follow-up are of an optimal standard. Accordingly, the authors conclude that the safety and efficiency of the proposed procedure for the therapeutic use of AM to treat various ocular pathologies are reproducible, with additional evidence favoring the use of AM as an alternative to conventional medical treatment for certain ocular conditions.

Analysis of the effectiveness of Sodium Hypochlorite decontamination of cadaveric human tissues at retrieval.

Bacterial contamination of tissues retrieved from cadaveric donors is a common feature worldwide, and every tissue bank, albeit using different methods, conducts decontamination to guarantee safe tissues suitable for clinical use. The effectiveness of the methods used to eradicate pathogens differs. In order to reduce the tissue bioburden at retrieval, we have introduced a new method involving rinsing tissues in a sodium hypochlorite solution. To test its effectiveness we analyzed two comparable groups of tissues: Group A: 1881 tissues, all rinsed with isotonic saline solution after retrieval, and Group B: 1968 tissues immersed in an isotonic saline solution containing sodium hypochlorite (final concentration 0.1 %) for different lengths of time and subsequently rinsed with isotonic saline. The rinsing solution of each tissue was then sampled for microbiological cultures in both groups. The resultant overall contamination rate was 40.5 % for Group A and 6.7 % for Group B, with an 82.8 % difference in the reduction of contamination between the two groups. This was especially the case for commensal skin bacteria in musculoskeletal tissue, which accounted for over half the overall contamination. Our data highlighted that decontamination with sodium hypochlorite was helpful in reducing the bacterial bioburden in tissues retrieved from cadaveric donors.

Evaluation of new antibiotic cocktails against contaminating bacteria found in allograft tissues.

Contamination of retrieved tissues is a major problem for allograft safety. Consequently, tissue banks have implemented decontamination protocols to eliminate microorganisms from tissues. Despite the widespread adoption of these protocols, few comprehensive studies validating such methods have been published. In this manuscript we compare the bactericidal activity of different antibiotic cocktails at different temperatures against a panel of bacterial species frequently isolated in allograft tissues collected at the Treviso Tissue Bank Foundation, a reference organization of the Veneto Region in Italy that was instituted to select, recover, process, store and distribute human tissues. We were able to identify at least two different formulations capable of killing most of the bacteria during prolonged incubation at 4 °C.

Extensive characterization of sphere models established from colorectal cancer cell lines.

Links between cancer and stem cells have been proposed for many years. As the cancer stem cell (CSC) theory became widely studied, new methods were developed to culture and expand cancer cells with conserved determinants of "stemness". These cells show increased ability to grow in suspension as spheres in serum-free medium supplemented with growth factors and chemicals. The physiological relevance of this phenomenon in established cancer cell lines remains unclear. Cell lines have traditionally been used to explore tumor biology and serve as preclinical models for the screening of potential therapeutic agents. Here, we grew cell-forming spheres (CFS) from 25 established colorectal cancer cell lines. The molecular and cellular characteristics of CFS were compared to the bulk of tumor cells. CFS could be isolated from 72 % of the cell lines. Both CFS and their parental CRC cell lines were highly tumorigenic. Compared to their parental cells, they showed similar expression of putative CSC markers. The ability of CRC cells to grow as CFS was greatly enhanced by prior treatment with 5-fluorouracil. At the molecular level, CFS and parental CRC cells showed identical gene mutations and very similar genomic profiles, although microarray analysis revealed changes in CFS gene expression that were independent of DNA copy-number. We identified a CFS gene expression signature common to CFS from all CRC cell lines, which was predictive of disease relapse in CRC patients. In conclusion, CFS models derived from CRC cell lines possess interesting phenotypic features that may have clinical relevance for drug resistance and disease relapse.

Three Decades of Experience with Aortic Prosthetic Valve Endocarditis.

The objective of this study was to evaluate early and long-term outcomes of patients with aortic prosthetic valve endocarditis (a-PVE) treated with a prosthetic aortic valve (PAV), prosthetic valved conduit (PVC), or cryopreserved aortic homograft (CAH). A total of 144 patients, 115 male and 29 female, aged 67 ± 12 years, underwent surgery for a-PVE at our institution between 1994 and 2021. Median time from the original cardiac surgery was 1.9 [0.6-5.6] years, and 47 (33%) patients developed an early a-PVE. Of these patients, 73 (51%) underwent aortic valve replacement (AVR) with a biological or mechanical PAV, 12 (8%) underwent aortic root replacement (ARR) with a biological or mechanical PVC, and 59 (42%) underwent AVR or ARR with a CAH. Patients treated with a CAH had significantly more circumferential annular abscess multiple valve involvement, longer CPB and aortic cross-clamping times, and needed more postoperative pacemaker implantation than patients treated with a PAV. No difference was observed in survival, reoperation rates, or recurrence of IE between patients treated with a PAV, a PVC, or a CAH. CAHs are technically more demanding and more often used in patients who have extensive annular abscess and multiple valve involvement. However, the use of CAH is safe in patients with complex a-PVE, and it shows excellent early and long-term outcomes.

A retrospective hospital benefit and cost analysis of the management of human tissues for orthopaedic allografts.

OBJECTIVES: The transplantation of human tissues is a greatly expanding field of medicine with unquestionable benefits that raise questions about safety, quality and ethics. Since 1 October 2019, the Fondazione Banca dei Tessuti del Veneto (FBTV) stopped sending thawed and ready to be transplanted cadaveric human tissues to hospitals. A retrospective analysis of the period 2016-2019 found a significant number of unused tissues. For this reason, the hospital pharmacy has developed a new centralised service characterised by thawing and washing human tissues for orthopaedic allografts. This study aims to analyse the hospital cost and benefit derived from this new service. METHODS: Aggregate data relating to tissue flows were obtained retrospectively for the period 2016-2022 through the hospital data warehouse. All tissues arriving from FBTV for each year were analysed, dividing them according to the outcome (if used or wasted). The percentage of wasted tissues as well as the economic loss due to wasted allografts were analysed per year and trimester. RESULTS: We identified 2484 allografts requested for the period 2016-2022. In the last 3 years of the analysis, characterised by the new tissue management of the pharmacy department, we found a statistically significant reduction in wasted tissues (p<0.0001) from 16.33% (216/1323) with a cost to the hospital of 176 866€ during the period 2016-2019 to 6.72% (78/1161) with a cost to the hospital of 79 423€ during the period 2020-2022. CONCLUSION: This study shows how the centralised processing of human tissues in the hospital pharmacy makes the procedure safer and more efficient, demonstrating how the synergy between different hospital departments, high professional skills and ethics can lead to a clinical advantage for patients and a better economic impact for the hospital.