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.

GCN5 contributes to intracellular lipid accumulation in human primary cardiac stromal cells from patients affected by Arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and cardiac fibro-fatty replacement. Over the last years, several works have demonstrated that different epigenetic enzymes can affect not only gene expression changes in cardiac diseases but also cellular metabolism. Specifically, the histone acetyltransferase GCN5 is known to facilitate adipogenesis and modulate cardiac metabolism in heart failure. Our group previously demonstrated that human primary cardiac stromal cells (CStCs) contribute to adipogenesis in the ACM pathology. Thus, this study aims to evaluate the role of GCN5 in ACM intracellular lipid accumulation. To do so, CStCs were obtained from right ventricle biopsies of ACM patients and from samples of healthy cadaveric donors (CTR). GCN5 expression was increased both in ex vivo and in vitro ACM samples compared to CTR. When GCN5 expression was silenced or pharmacologically inhibited by the administration of MB-3, we observed a reduction in lipid accumulation and a mitigation of reactive oxygen species (ROS) production in ACM CStCs. In agreement, transcriptome analysis revealed that the presence of MB-3 modified the expression of pathways related to cellular redox balance. Altogether, our findings suggest that GCN5 inhibition reduces fat accumulation in ACM CStCs, partially by modulating intracellular redox balance pathways.

Oxidized LDL-dependent pathway as new pathogenic trigger in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is hallmarked by ventricular fibro-adipogenic alterations, contributing to cardiac dysfunctions and arrhythmias. Although genetically determined (e.g., PKP2 mutations), ACM phenotypes are highly variable. More data on phenotype modulators, clinical prognosticators, and etiological therapies are awaited. We hypothesized that oxidized low-density lipoprotein (oxLDL)-dependent activation of PPARγ, a recognized effector of ACM adipogenesis, contributes to disease pathogenesis. ACM patients showing high plasma concentration of oxLDL display severe clinical phenotypes in terms of fat infiltration, ventricular dysfunction, and major arrhythmic event risk. In ACM patient-derived cardiac cells, we demonstrated that oxLDLs are major cofactors of adipogenesis. Mechanistically, the increased lipid accumulation is mediated by oxLDL cell internalization through CD36, ultimately resulting in PPARγ upregulation. By boosting oxLDL in a Pkp2 heterozygous knock-out mice through high-fat diet feeding, we confirmed in vivo the oxidized lipid dependency of cardiac adipogenesis and right ventricle systolic impairment, which are counteracted by atorvastatin treatment. The modulatory role of oxidized lipids on ACM adipogenesis, demonstrated at cellular, mouse, and patient levels, represents a novel risk stratification tool and a target for ACM pharmacological strategies.

Differences in Mitochondrial Membrane Potential Identify Distinct Populations of Human Cardiac Mesenchymal Progenitor Cells.

Adult human cardiac mesenchymal progenitor cells (hCmPC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Even if the mechanisms have not yet been fully elucidated, the stem cell differentiation is guided by the mitochondrial metabolism; however, mitochondrial approaches to identify hCmPC with enhanced stemness and/or differentiation capability for cellular therapy are not established. Here we demonstrated that hCmPCs sorted for low and high mitochondrial membrane potential (using a lipophilic cationic dye tetramethylrhodamine methyl ester, TMRM), presented differences in energy metabolism from preferential glycolysis to oxidative rates. TMRM-high cells are highly efficient in terms of oxygen consumption rate, basal and maximal respiration, and spare respiratory capacity compared to TMRM-low cells. TMRM-high cells showed characteristics of pre-committed cells and were associated with higher in vitro differentiation capacity through endothelial, cardiac-like, and, to a lesser extent, adipogenic and chondro/osteogenic cell lineage, when compared with TMRM-low cells. Conversely, TMRM-low showed higher self-renewal potential. To conclude, we identified two hCmPC populations with different metabolic profile, stemness maturity, and differentiation potential. Our findings suggest that metabolic sorting can isolate cells with higher regenerative capacity and/or long-term survival. This metabolism-based strategy to select cells may be broadly applicable to therapies.

Cyclophilin A/EMMPRIN Axis Is Involved in Pro-Fibrotic Processes Associated with Thoracic Aortic Aneurysm of Marfan Syndrome Patients.

: Background: Marfan syndrome (MFS) is a genetic disease, characterized by thoracic aortic aneurysm (TAA), which treatment is to date purely surgical. Understanding of novel molecular targets is mandatory to unveil effective pharmacological approaches. Cyclophilin A (CyPA) and its receptor EMMPRIN are associated with several cardiovascular diseases, including abdominal aortic aneurysm. Here, we envisioned the contribution of CyPA/EMMPRIN axis in MFS-related TAA. METHODS: We obtained thoracic aortic samples from healthy controls (HC) and MFS patients' aortas and then isolated vascular smooth muscle cells (VSMC) from the aortic wall. RESULTS: our findings revealed that MFS aortic tissue samples isolated from the dilated zone of aorta showed higher expression levels of EMMPRIN vs. MFS non-dilated aorta and HC. Interestingly, angiotensin II significantly stimulated CyPA secretion in MFS-derived VSMC (MFS-VSMC). CyPA treatment on MFS-VSMC led to increased levels of EMMPRIN and other MFS-associated pro-fibrotic mediators, such as TGF-ß1 and collagen I. These molecules were downregulated by in vitro treatment with CyPA inhibitor MM284. Our results suggest that CyPA/EMMPRIN axis is involved in MFS-related TAA development, since EMMPRIN is upregulated in the dilated zone of MFS patients' TAA and the inhibition of its ligand, CyPA, downregulated EMMPRIN and MFS-related markers in MFS-VSMC. CONCLUSIONS: these insights suggest both a novel detrimental role for CyPA/EMMPRIN axis and its inhibition as a potential therapeutic strategy for MFS-related TAA treatment.

Cyclophilin A in Arrhythmogenic Cardiomyopathy Cardiac Remodeling.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder characterized by the progressive substitution of functional myocardium with noncontractile fibro-fatty tissue contributing to ventricular arrhythmias and sudden cardiac death. Cyclophilin A (CyPA) is a ubiquitous protein involved in several pathological mechanisms, which also characterize ACM (i.e., fibrosis, inflammation, and adipogenesis). Nevertheless, the involvement of CyPA in ACM cardiac remodeling has not been investigated yet. Thus, we first evaluated CyPA expression levels in the right ventricle (RV) tissue specimens obtained from ACM patients and healthy controls (HC) by immunohistochemistry. Then, we took advantage of ACM- and HC-derived cardiac mesenchymal stromal cells (C-MSC) to assess CyPA modulation during adipogenic differentiation. Interestingly, CyPA was more expressed in the RV sections obtained from ACM vs. HC subjects and positively correlated with the adipose replacement extent. Moreover, CyPA was upregulated at early stages of C-MSC adipogenic differentiation and was secreted at higher level over time in ACM- derived C-MSC. Our study provides novel ex vivo and in vitro information on CyPA expression in ACM remodeling paving the way for future C-MSC-based mechanistic and therapeutic investigations.

Human Amniotic Membrane Positioning in the Surgical Treatment of Temporomandibular Joint Degenerative Disorder.

BACKGROUND: Temporomandibular joint (TMJ) arthritis is a degenerative pathology that may cause pain and dysfunction. Nonsurgical therapy is the traditional treatment of TMJ diseases but if ineffective, TMJ surgery can be performed and may include arthroplasty with interposition of autograft. The encouraging results reported with the use of human amniotic membrane (HAM) in different surgical fields have highlighted its potential, but approaches providing the positioning of HAM within the intra-articular space of arthritic TMJs have never been investigated. CASE PRESENTATION: A 48-year-old woman was presented with limited mouth opening and pain with palpation at the left joint. A severe TMJ degeneration was diagnosed, and a surgical treatment was necessary. In the present case report, the authors describe the application of a cryopreserved HAM patch within the joint space as a disc-replacing film during major surgeries for discectomy and arthroplasty. Three months after the intervention, the patient reported an overall improvement in chewing efficiency as well as the absence of pain. CONCLUSIONS: According to the regenerative effects of HAM, the design of trials on the topic should be encouraged for its possible inclusion within the field of TMJ disease practice.

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.

The arrhythmogenic cardiomyopathy-specific coding and non-coding transcriptome in human cardiac stromal cells.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is a genetic autosomal disease characterized by abnormal cell-cell adhesion, cardiomyocyte death, progressive fibro-adipose replacement of the myocardium, arrhythmias and sudden death. Several different cell types contribute to the pathogenesis of ACM, including, as recently described, cardiac stromal cells (CStCs). In the present study, we aim to identify ACM-specific expression profiles of human CStCs derived from endomyocardial biopsies of ACM patients and healthy individuals employing TaqMan Low Density Arrays for miRNA expression profiling, and high throughput sequencing for gene expression quantification. RESULTS: We identified 3 miRNAs and 272 genes as significantly differentially expressed at a 5% false discovery rate. Both the differentially expressed genes as well as the target genes of the ACM-specific miRNAs were found to be enriched in cell adhesion-related biological processes. Functional similarity and protein interaction-based network analyses performed on the identified deregulated genes, miRNA targets and known ACM-causative genes revealed clusters of highly related genes involved in cell adhesion, extracellular matrix organization, lipid transport and ephrin receptor signaling. CONCLUSIONS: We determined for the first time the coding and non-coding transcriptome characteristic of ACM cardiac stromal cells, finding evidence for a potential contribution of miRNAs, specifically miR-29b-3p, to ACM pathogenesis or phenotype maintenance.

Decellularized Cryopreserved Allografts as Off-the-Shelf Allogeneic Alternative for Heart Valve Replacement: In Vitro Assessment Before Clinical Translation.

Cryopreserved allogeneic conduits are the elective biocompatible choice among currently available substitutes for surgical replacement in end-stage valvulopathy. However, degeneration occurs in 15 years in adults or faster in children, due to recipient's immunological reactions to donor's antigens. Here, human aortic valves were decellularized by TRICOL, based on Triton X-100 and sodium cholate, and submitted to standard cryopreservation (TRICOL-human aortic valves (hAVs)). Tissue samples were analyzed to study the effects of the combined procedure on original valve architecture and donor's cell removal. Residual amounts of nucleic acids, pathological microorganisms, and detergents were also investigated. TRICOL-hAVs proved to be efficaciously decellularized with removal of donor's cell components and preservation of valve scaffolding. Trivial traces of detergents, no cytotoxicity, and abrogated bioburden were documented. TRICOL-hAVs may represent off-the-shelf alternatives for both aortic and pulmonary valve replacements in pediatric and grown-up with congenital heart disease patients.

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.

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.

Nerve regeneration across cryopreserved allografts from cadaveric donors: a novel approach for peripheral nerve reconstruction.

OBJECT: The use of allografts from cadaveric donors has attracted renewed interest in recent years, and pretreatment with cryopreservation and immunosuppression methods has been investigated to maximize axonal regrowth and minimize allograft rejection. The authors wanted to assess the outcome of treatments of brachial plexus stretch injuries with cryopreserved allografts from cadaveric donors in nonimmunosuppressed patients. METHODS: Ten patients with brachial plexus lesions were submitted to electromyography (EMG) testing 1 and 3 months after a traumatic event and 1 week before surgery to localize and identify the type of lesion. Intraoperative EMG recordings were performed for intraoperative monitoring to select the best surgical strategy, and postoperative EMG was used to follow up patients and determine surgical outcomes. If nerve action potentials (NAPs) were present intraoperatively, neurolysis was performed, whereas muscular/nerve neurotization was performed if NAPs were absent. Cryopreserved allografts obtained from selected cadaveric donors and provided by the tissue bank of Treviso were used for nerve reconstruction in patients who were not treated with immunosuppressive drugs. RESULTS: The surgical strategy was selected according to the type and site of the nerve lesion and on the basis of IOM results: 14 cryopreserved allografts were used for 7 muscular neurotizations and for 7 nerve neurotizations, and 5 neurolysis procedures were performed. All of the patients had regained motor function at the 1- and 2-year follow-ups. CONCLUSIONS: Some variables may affect functional recovery after allograft surgery, and the outcome of peripheral nerve reconstruction is more favorable when patients are carefully evaluated and selected for the surgery. The authors demonstrated that using cryopreserved allografts from cadaveric donors is a valid surgical strategy to restore function of the damaged nerve without the need for any immunosuppressive treatments. This approach offers new perspectives on procedures for extensive reconstruction of brachial and lumbosacral plexuses.

Effectiveness of fresh frozen and cryopreserved homologue iliac crest grafts used in sinus lifting: a comparative study.

In the last decade, several investigators have reported that autologous and homologous fresh frozen bones (FFB) are effective materials to restore alveolar ridges previous to insert dental implants. Recently we have used cryopreserved homologue grafts (CFFB). Here we reported a retrospective comparative study between implants inserted in FFB and CFFB evaluate their clinical outcome. Patients were treated with a split mouth scheme for bone grafting with FFB and CFFB and spiral family implants (SPI) were inserted in the same surgical time. Several variables (patient, grafts, anatomic site, implant, prosthetic restoration) were investigated. Implant' failure and peri-implant bone resorption were considered as predictor of clinical outcome. A total of 84 SFIs were inserted in 12 patients. Implants were inserted to replace 8 incisors, 4 cuspids, 31 premolars and 41 molars. The mean follow-up was 14 months. Three out of 84 implants was lost (i.e. survival rate SVR = 96.4%) and no differences were detected among the studied variables. Similar result was obtained by analyzing the crestal bone resorption around implant' neck (i.e. success rate). FFB and CFFB have high and comparable survival and success rate. Implants inserted with one step surgical procedure in native (i.e. not grafted) bone, FFB and CFFB have similar clinical outcome.