Renal Vessel Extension With Cryopreserved Vascular Grafts: Overcoming Surgical Pitfalls in Living Donor Kidney Transplant.

In LDKT, right kidneys and kidneys with anomalous vascularization are often deferred because of concerns on complications and vascular reconstructions. To date, only few reports have examined renal vessel extension with cryopreserved vascular grafts in LDKT. The aim of this study is to investigate the effect of renal vessel extension on short-term outcomes and ischemia times in LDKT. From 2012 to 2020, recipients of LDKT with renal vessels extension were compared with standard LDKT recipients. Subset analysis of rights grafts and grafts with anomalous vascularization, with or without renal vessel extension, was performed. Recipients of LDKT with (n = 54) and without (n = 91) vascular extension experienced similar hospital stays, surgical complications and DGF rates. For grafts with multiple vessels, renal vessel extension granted a faster implantation time (44±5 vs. 72±14 min), which resulted comparable to that of standard anatomy grafts. Right kidney grafts with vascular extension had a faster implantation time compared to right kidney grafts without vascular lengthening (43±5 vs. 58±9 min), and a comparable implantation time to left kidney grafts. Renal vessel extension with cryopreserved vascular grafts allows faster implantation time in right kidney grafts or grafts with anomalous vascularization, maintaining similar surgical and functional outcomes.

Impact of Freeze-Drying on Cord Blood (CB), Serum (S), and Platelet-Rich Plasma (CB-PRP) Preparations on Growth Factor Content and In Vitro Cell Wound Healing.

Blood-based preparations are used in clinical practice for the treatment of several eye disorders. The aim of this study is to analyze the effect of freeze-drying blood-based preparations on the levels of growth factors and wound healing behaviors in an in vitro model. Platelet-rich plasma (PRP) and serum (S) preparations from the same Cord Blood (CB) sample, prepared in both fresh frozen (FF) and freeze-dried (FD) forms (and then reconstituted), were analyzed for EGF and BDNF content (ELISA Quantikine kit). The human MIO-M1 glial cell line (Moorfield/Institute of Ophthalmology, London, UK) was incubated with FF and FD products and evaluated for cell migration with scratch-induced wounding (IncuCyte S3 Essen BioScience), proliferation with cyclin A2 and D1 gene expression, and activation with vimentin and GFAP gene expression. The FF and FD forms showed similar concentrations of EGF and BDNF in both the S and PRP preparations. The wound healing assay showed no significant difference between the FF and FD forms for both S and PRP. Additionally, cell migration, proliferation, and activation did not appear to change in the FD forms compared to the FF ones. Our study showed that reconstituted FD products maintained the growth factor concentrations and biological properties of FF products and could be used as a functional treatment option.

Third-party bone marrow-derived mesenchymal stromal cell infusion before liver transplantation: A randomized controlled trial.

Mesenchymal stromal cells (MSC) have emerged as a promising therapy to minimize the immunosuppressive regimen or induce tolerance in solid organ transplantation. In this randomized open-label phase Ib/IIa clinical trial, 20 liver transplant patients were randomly allocated (1:1) to receive a single pretransplant intravenous infusion of third-party bone marrow-derived MSC or standard of care alone. The primary endpoint was the safety profile of MSC administration during the 1-year follow-up. In all, 19 patients completed the study, and none of those who received MSC experienced infusion-related complications. The incidence of serious and non-serious adverse events was similar in the two groups. Circulating Treg/memory Treg and tolerant NK subset of CD56bright NK cells increased slightly over baseline, albeit not to a statistically significant extent, in MSC-treated patients but not in the control group. Graft function and survival, as well as histologic parameters and intragraft expression of tolerance-associated transcripts in 1-year protocol biopsies were similar in the two groups. In conclusion, pretransplant MSC infusion in liver transplant recipients was safe and induced mild positive changes in immunoregulatory T and NK cells in the peripheral blood. This study opens the way for a trial on possible tolerogenic efficacy of MSC in liver transplantation. ClinicalTrials.gov identifier: NCT02260375.

Metabolomics comparison of cord and peripheral blood-derived serum eye drops for the treatment of dry eye disease.

Allogeneic peripheral blood-derived (PBS) serum eye drops have been largely used in the treatment of dry eye disease (DED). Recently, cord blood has emerged as an effective alternative serum source (cord blood serum, CBS), containing a higher amount of growth factors than PBS, it holds the promise of a better capability to stimulate corneal healing. However, the lack of a standardized method for preparation, dispensation, storage and a poor biochemical characterization still hamper the establishment of a clinical consensus. Here the metabolomes of the two different serum eye drop preparations were compared using proton nuclear magnetic resonance spectroscopy. We found that both PBS and CBS contained several organic compounds, the majority of them already detected in human tears and may be thereby considered lacrimal substitutes. Metabolites having in the multivariate statistical analysis Partial least squares discriminant analysis (PLS-DA) a VIP scores > 1.0 were considered to be significantly different. All the metabolites identified were found to have a p < 0.05 in the univariate analysis. CBS, in particular, showed the highest amount of choline, myo-inositol, glutamine, creatine and ß-hydroxybutyrate. These evidences constitute relevant advances towards serum eye drops characterization and confirm that cord blood is a valid alternative source of serum eye drops.

Blood derived eye drops for the treatment of cornea and ocular surface diseases.

The use of blood derived eye drops for the treatment of ocular surface disorders has become increasingly popular in recent years. The mechanism of action is the stimulation of cellular proliferation and migration by supplying an active mixture of growth factors and cytokines at the ocular surface, thus mimicking the function of the lacking natural tears. Blood derived eye drops have been used in the last decades for the treatment of a variety of ocular surface diseases, including mainly dry eye disease, persistent corneal epithelial defect, corneal ulcer, ocular surface burn, recurrent corneal erosion and limbal stem-cell deficiency. Among overall blood derived eye drops, both autologous (from the patients themselves) and homologous (from donors) products exist, with different advantages and disadvantages. Autologous serum, obtained from the patient's own peripheral blood, is the first introduced and most commonly used product. Despite several randomized clinical trials showed its safety and efficacy, a recent Cochraine meta-analysis failed to show significant results due to low evidence. Homologous sources including allogeneic serum obtained from healthy donors, and umbilical cord blood serum collected at the time of delivery, are efficient alternatives, especially when autologous serum therapy is contraindicated or not appropriate. Platelet-derived eye drops are prepared and used in various but poor standardized preparations, namely platelet-rich plasma, plasma rich in growth factors, and platelet lysate. Future perspectives of blood-derived products include the introduction of tailored eye drops, screened for the proper content of growth factors and cytokines according to each patient and ocular surface disease.

The incidence and morphology of Monckeberg's medial calcification in banked vascular segments from a monocentric donor population.

Little is known about the real incidence and the clinical relevance of the enigmatic Monckeberg's medial calcification in the patency of the femoral artery allograft. Here we present a retrospective study on 143 multiorgan donors (mean age 38 years, range 14-59 years), to describe the incidence and the morphological features of vascular calcifications in banked femoral arteries suitable for clinical use. In the present series, focal vascular calcifications were present in 36 (25 %) cases, 23 cases localized in the intima, 7 in the media, and 6 were mixed. No correlation was found between the incidence of calcifications and the classical cardiovascular clinical risk factors (n = 9); only hypertension correlated with the medial localization, but not with the incidence, of the calcification (P = 0.017). While the macroscopic exclusion criteria of vascular grafts include atheromatous and not-atheromatous lesions, we ignore the actual impact of Monckeberg's medial calcification on vessel transplantation and allograft life. In our opinion this is a very important topic, since when the histological criteria for Monckeberg's calcification diagnosis are used, 25 % of our young donors population was affected. Whether Monckeberg's medial calcification is a stable arterial condition, apparently underestimated in the general population, or a dynamic process evolving with age and atherosclerosis, or a banking-related vascular alteration, still remain an open issue deserving further studies with subjects of different ages.

Human Vascular Wall Mesenchymal Stromal Cells Contribute to Abdominal Aortic Aneurysm Pathogenesis Through an Impaired Immunomodulatory Activity and Increased Levels of Matrix Metalloproteinase-9.

BACKGROUND: The main histopathological features of abdominal aortic aneurysm (AAA) are tissue proteolysis mediated by matrix metalloproteinases (MMPs) and inflammation. This study aimed at verifying the presence and contribution of mesenchymal stromal cells (MSCs) to aneurysmal tissue remodeling. METHODS AND RESULTS: MSCs were successfully isolated from the AAA wall of 12 male patients and were found to express mesenchymal and stemness markers. MMP-2/-9 are involved in AAA progression and their mRNA levels in AAA-MSCs resulted higher than healthy MSCs (cMSCs), especially MMP-9 (400-fold increased). Moreover, MMP-9 protein and activity were pronounced in AAA-MSCs. Immunomodulation was tested in AAA-MSCs after co-culture with activated peripheral blood mononuclear cells (PBMCs) and revealed a weak immunosuppressive action on PBMC proliferation (bromodeoxyuridine incorporation, flow cytometry assay), together with a reduced expression of anti-inflammatory molecules (HLA-G, IL-10) by AAA-MSCs compared to cMSCs. MMP-9 expression in AAA-MSCs was shown to be negatively modulated under the influence of cMSCs and exogenous IL-10. CONCLUSIONS: MSCs with stemness properties are niched in human AAA tissues and display a dysregulation of functional activities; that is, upregulation of MMP-9 and ineffective immunomodulatory capacity, which are crucial in the AAA progression; the possibility to modulate the increased MMP-9 expression by healthy MSCs and IL-10 suggests that novel therapeutic strategies are possible for slowing down AAA progression.

Topical applications of heterologous platelet rich plasma (PRP) for refractory gingival lesions in autoimmune blistering diseases.

BACKGROUND: Recalcitrant gingival erosions, blisters and desquamative gingivitis are common features in oral autoimmune blistering diseases (AIBD). First line treatments include high-dosage corticosteroids and other immunosuppressive drugs, with several side effects and elevated number of recurrences. Autologous platelet-rich plasma (PRP) has been recently introduced as an alternative treatment and its use seems to be promising and safe. METHODS: In this study we describe the use of topical application of heterologous PRP in nine patients affected by mucous membrane pemphigoid, with gingival lesions refractory to previous treatments. Topical applications of PRP were performed once a week for 2 months and the endpoint for clinical evaluation was set 3 months after the last session. Oral disease severity score (ODSS) and VAS scores for pain measurement were recorded before and after treatment. RESULTS: The procedure was painless, well accepted, and free from adverse reactions. All patients (100%) reported a reduction in VAS whereas reduction in ODSS was observed in 89% of patients. CONCLUSIONS: Within the limits of the study, topical heterologous PRP is a safe and promising procedure to be studied in future controlled randomized trials as adjuvant treatment for refractory gingival lesions in patients with AIBDs.

Platelet-rich plasma therapy for postoperative esophageal fistula in a pediatric patient.

Postoperative management of esophagocutaneous fistulas in pediatric patients is challenging, often resulting in prolonged hospitalization and increased morbidity. Platelet-rich plasma (PRP) has emerged as a promising adjunctive treatment for such complications. We present the case of a 7-month-old infant who developed an esophago-cutaneous fistula following esophagocoloplasty for esophageal atresia type A. Despite initial conservative management, the fistula persisted, prompting the application of PRP gel derived from umbilical cord blood. After four applications of PRP, complete closure of the fistula was achieved, leading to both functional and aesthetic results. This case highlights the potential of PRP in managing refractory postoperative esophageal fistulas in pediatric patients and underscores the need for further research to optimize treatment protocols and validate its efficacy for this sort of complications.

Establishing a procedure for dimethyl sulfoxide removal from cardiovascular allografts: a quantitative study.

The success of allotransplants is critically dependent on both tissue viability and efficient removal of potentially toxic cryopreservants. In this study we analysed the dimethyl sulphoxide (Me2SO) content of cardiovascular tissue samples stored in tissue banks and optimized a washing protocol to be used before surgical implant. We compared the Me2SO content of heart valves, arteries and veins and quantitatively determined by HPLC the washing kinetics of each group of tissue samples under strictly controlled conditions using an industrial washing medium (BASE). Our results showed that heart valves and arteries have significantly slower Me2SO release kinetics than veins. Approximately 20 % of the initial content of cryopreservant could still be detected in the valves and arterial tissue after 15 min of continuous washing. Conversely, veins were almost completely cleared of the cryoprotectant under the same conditions. We propose a washing protocol consisting of two sequential washing with BASE for a total of 25 min for valves and arteries and 15 min for veins. In our hands, this protocol reliably ensures the removal of more than 95 % of the initial Me2SO content.

Phenotypic, morphological, and metabolic characterization of vascular-spheres from human vascular mesenchymal stem cells.

The ability to form spheroids under non-adherent conditions is a well-known property of human mesenchymal stem cells (hMSCs), in addition to stemness and multilineage differentiation features. In the present study, we tested the ability of hMSCs isolated from the vascular wall (hVW-MSCs) to grow as spheres, and provide a characterization of this 3D model. hVW-MSCs were isolated from femoral arteries through enzymatic digestion. Spheres were obtained using ultra-low attachment and hanging drop methods. Immunophenotype and pluripotent genes (SOX-2, OCT-4, NANOG) were analyzed by immunocytochemistry and real-time PCR, respectively. Spheres histological and ultrastructural architecture were examined. Cell viability and proliferative capacity were measured using LIVE/DEATH assay and ki-67 proliferation marker. Metabolomic profile was obtained with liquid chromatography-mass spectrometry. In 2D, hVW-MSCs were spindle-shaped, expressed mesenchymal antigens, and displayed mesengenic potential. 3D cultures of hVW-MSCs were CD44+ , CD105low , CD90low , exhibited a low propensity to enter the cell cycle as indicated by low percentage of ki-67 expression and accumulated intermediate metabolites pointing to slowed metabolism. The 3D model of hVW-MSCs exhibits stemness, dormancy and slow metabolism, typically observed in stem cell niches. This culture strategy can represent an accurate model to investigate hMSCs features for future clinical applications in the vascular field.

Impact of blood source and component manufacturing on neurotrophin content and in vitro cell wound healing.

BACKGROUND: We evaluated neurotrophin (NF) levels and their impact on in vitro cell wound healing in eye drops from differently prepared blood sources (cord blood [CB], and peripheral blood [PB]) in the same donor, to avoid intrasubject biological variability. MATERIALS AND METHODS: Twenty healthy adult donor PB samples, and twenty CB samples acquired at the time of delivery were processed to obtain serum (S), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and S retrieved from PRP after activation with Ca-gluconate (PRP-R). The levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) were assessed with a Luminex xMAP (Luminex Corporation), and by using multikine kits from R&D system, and were statistically analysed in the eight different preparations. The impact of S, PRP, PPP, PRP-R from both sources on a cell line responding to NF supplementation (MIO-M1, UCL Institute of Ophthalmology, London, UK) was tested with a scratch wound assay, and analysed by IncuCyte S3 equipment. RESULTS: All the preparations from CB showed higher NF levels, except for BDNF where no difference was found as compared to PB. PRP showed higher NF levels with respect to S, PPP and PRP-R in this decreasing order. Younger donors in PB contributed with higher NF levels. The scratch assay showed different cell migration results, with a complete wound closure only recorded with the supplementation of CB-S, and a progressive reduction by using PRP, PRP-R, and PPP from both sources. DISCUSSION: Protocols of preparation and choice of blood source determine different NF levels in the final products. The therapeutic use of a natural neurotrophin pool from blood sources might have a clinical impact in several different settings. Efforts are needed to standardise the manufacturing and the product content in order to establish and modulate the posology of the final supplementation.

Umbilical Cord PRP vs. Autologous PRP for the Treatment of Hip Osteoarthritis.

Umbilical cord platelet-rich plasma (C-PRP) has more growth factors and anti-inflammatory molecules compared with autologous PRP (A-PRP) derived from peripheral blood. The aim of this study was to compare intra-articular C-PRP or A-PRP injections in terms of safety and clinical efficacy for the treatment of patients with hip osteoarthritis (OA). This study investigated the results of 100 patients with hip OA treated with three weekly ultrasound-guided injections of either C-PRP or A-PRP. Clinical evaluations were performed before the treatment and after two, six, and twelve months with the HHS, WOMAC, and VAS scores. No major adverse events were recorded. Overall, the improvement was limited with both treatments. Significant improvements in VAS (p = 0.031) and HHS (p = 0.011) were documented at two months for C-PRP. Patients with a low OA grade (Tonnis 1-2) showed a significantly higher HHS improvement with C-PRP than A-PRP at twelve months (p = 0.049). C-PRP injections are safe but offered only a short-term clinical improvement. The comparative analysis did not demonstrate benefits compared with A-PRP in the overall population, but the results are influenced by OA severity, with C-PRP showing more benefits when advanced OA cases were excluded. Further studies are needed to confirm the most suitable indications and potential of this biological injective approach.

Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics.

BACKGROUND AIMS: The presence of ectopic tissues in the pathologic artery wall raises the issue of whether multipotent stem cells may reside in the vasculature itself. Recently mesenchymal stromal cells (MSC) have been isolated from different human vascular segments (VW MSC), belying the previous view that the vessel wall is a relatively quiescent tissue. METHODS: Resident multipotent cells were recovered from fresh arterial segments (aortic arches, thoracic and femoral arteries) collected in a tissue-banking facility and used to establish an in situ and in vitro study of the stemness features and multipotency of these multidistrict MSC populations. RESULTS: Notch-1+, Stro-1+, Sca-1+ and Oct-4+ cells were distributed along an arterial wall vasculogenic niche. Multidistrict VW MSC homogeneously expressed markers of stemness (Stro-1, Notch-1 and Oct-4) and MSC lineages (CD44, CD90, CD105, CD73, CD29 and CD166) whilst they were negative for hematopoietic and endothelial markers (CD34, CD45, CD31 and vWF). Each VW MSC population had characteristics of stem cells, i.e. a high efflux capability for Hoechst 33342 dye and the ability to form spheroids when grown in suspension and generate colonies when seeded at low density. Again, VW MSC cultured in induction media exhibited adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic differentiation, as documented by histochemical, immunohistochemical, molecular and electron microscopy analysis. CONCLUSIONS: Overall, these findings may enlighten the physiopathologic mechanisms of vascular wall diseases as well as having potential implications for cellular, genetic and tissue engineering approaches to treating vascular pathologies when these are unresponsive to medical and surgical therapies.

Cord Blood Serum (CBS)-Based Eye Drops Modulate Light-Induced Neurodegeneration in Albino Rat Retinas.

Age-related macular degeneration (AMD) is one of the leading causes of visual loss in western countries, it has no cure, and its incidence will grow in the future, for the overall population aging. Albino rats with retinal degeneration induced by exposure to high-intensity light (light-damage, LD) have been extensively used as a model of AMD to test neuroprotective agents. Among them, trophic factors (NGF and BDNF) have been shown to play a significant role in photoreceptors' survival. Interestingly, cord blood serum (CBS) is an extract full of chemokines and trophic factors; we, therefore, hypothesized that CBS could be an excellent candidate for neuroprotection. Here, we investigate whether CBS-based eye drops might mitigate the effects of light-induced retinal degeneration in albino rats. CBS treatment significantly preserved flash-electroretinogram (f-ERG) response after LD and reduced the "hot-spot" extension. Besides, CBS-treated animals better preserved the morphology of the outer nuclear layer, together with a reduction in microglia migration and activation. Interestingly, the treatment did not modulate reactive gliosis and activation of the self-protective mechanism (FGF2). In conclusion, our results suggest that CBS-based eye drops might be successfully used to mitigate retinal neurodegenerative processes such as AMD.

Effects of Cord Blood Serum (CBS) on viability of retinal Müller glial cells under in vitro injury.

Oxidative stress and inflammation determine retinal ganglion cell degeneration, leading to retinal impairment and vision loss. Müller glial cells regulate retinal repair under injury, through gliosis. Meanwhile, reactive gliosis can turn in pathological effects, contributing to neurodegeneration. In the present study, we tested whether Cord Blood Serum (CBS), rich of growth factors, might improve the viability of Müller cells under in vitro damage. BDNF, NGF, TGF-α, GDNF and EGF levels were measured in CBS samples by Human Magnetic Luminex Assay. CBS effects were evaluated on rat (rMC-1) and human (MIO-M1) Müller cells, under H2O2 and IL-1ß damage. Cells grown with FBS or CBS both at 5% were exposed to stress and analyzed in terms of cell viability, GFAP, IL-6 and TNF-α expression. CBS was also administrated after treatment with K252a, inhibitor of the neurotrophin receptor Trk. Cell viability of rMC-1 and MIO-M1 resulted significantly improved when pretreated with CBS and exposed to H2O2 and IL-1ß, in comparison to the standard culture with FBS. Accordingly, the gliosis marker GFAP resulted down-regulated following CBS priming. In parallel, we observed a lower expression of the inflammatory mediators in rMC-1 (TNF-α) and MIO-M1 (IL-6, TNF- α), especially in presence of inflammatory damage. Trk inhibition through K252a administration impaired the effects of CBS under stress conditions on MIO-M1 and rMC-1 viability, not significantly different from FBS condition. CBS is enriched with neurotrophins and its administration to rMC-1 and MIO-M1 attenuates the cytotoxic effects of H2O2 and IL-1ß. Moreover, the decrease of the main markers of gliosis and inflammation suggests a promising use of CBS for neuroprotection aims. This study is a preliminary basis that prompts future investigations to deeply explore and confirm the CBS potential.

"Mom Let's Go to the Dentist!" Preliminary Feasibility of a Tailored Dental Intervention for Children with Autism Spectrum Disorder in the Italian Public Health Service.

Children with autism spectrum disorder (ASD) show worse oral health than their peers. Their access to health services is, at present, inadequate: few high-quality interventions have been designed and implemented to improve their care procedures so far. The purpose of this study is to describe an experience of dental care supported by Information and Communication Technologies (ICT), for children with ASD in a public health service. In our study, 59 children (mean age 9.9 years; SD = 5.43) participated in the MyDentist project. It integrates classic dental care techniques with new practices for desensitization and fear control, delivered through an enhanced customized ICT-based intervention aiming at familiarizing the child with ASD with the medical setting and procedures. Two questionnaires were filled out by parents to describe the acceptability of the MyDentist experience for their children. Significant results were shown from T0 (before initiating MyDentist) to T1 (after 6 months of the MyDentist experience) regarding improved oral hygiene and cooperation during dental treatments. Families positively assessed the use of ICT support. In conclusion, the project demonstrated acceptability by parents, suggesting that public health dental care and prevention can be successfully implemented without resorting to costly pharmacological interventions (with potential side effects), taking better care of children's health.

Blood derived treatment from two allogeneic sources for severe dry eye associated to keratopathy: a multicentre randomised cross over clinical trial.

AIM: To compare the efficacy of cord blood and peripheral adult donor blood serum eyedrops, controlled for growth factor content, in the treatment of severe dry eye diseases (DED) resistant to conventional therapy. METHODS: This was a multicentre randomised, double-masked, cross-over clinical trial. Sixty patients diagnosed as severe DED, associated to persistent corneal epithelial defects were randomised and equally assigned to group A (treated with cord blood serum (CBS)) or group B (treated with PBS), eyedrops administered eight times/day for 1 month. Primary outcome was the pretreatment and post-treatment change in corneal fluorescein staining. Secondary outcomes included the pretreatment and post-treatment change in Ocular Surface Disease Index (OSDI) questionnaire and Visual Analogue Score (VAS) of subjective symptoms, Schirmer I test, tear break-up time and conjunctival staining. Patients with relapse in signs or symptoms after further 2 months switched to the remaining group for one additional month. Data were statistically analysed (p<0.05). RESULTS: Corneal staining was more significantly reduced after the CBS treatment, both VAS and OSDI score reduction was observed in both groups, but group A reported significantly less grittiness and pain. Nineteen patients shifted in the crossover period, the within individual comparison confirmed a better recovery in the CBS treatment period. Reduction in epithelial damage was positively associated with epidermal growth factor, transforming growth factorα and platelet-derived growth factor content. Levels of interleukins (IL-13) were positively associated with symptom decrease. CONCLUSIONS: Overall, DED signs improved after both CBS and PBS treatments, with potential advantages of CBS for subjective symptoms and corneal damage reduction. CLINICAL TRIAL REGISTRATION: NCT03064984.

Blood-Based Treatments for Severe Dry Eye Disease: The Need of a Consensus.

The use of blood-based eye drops as therapy for various diseases of the ocular surface has become increasingly popular in ophthalmic practice during recent years. The rationale for their use is based on the promotion of cellular proliferation and migration thanks to the supply of metabolically active substances, in particular growth factors. Blood-derived eye drops have been used for the treatment of several ocular surface disorders, such as dry eye disease, corneal ulcer, persistent epithelial defect, neurotrophic keratitis, ocular surface burn, recurrent corneal erosion, and limbal stem-cell deficiency. Both autologous (from patients themselves) and heterologous (from adult donors or from cord blood sampled at birth)-derived products exist, and each source has specific pros and cons. Despite an extensive literature, several issues are still under debate and the aim of this manuscript is to review the indications, preparation methods and storage, characterization of content, rationale for clinical outcomes, patient stratification, length of treatment, and rationale for repeated treatments at disease relapse. A rationale based on a "5 Ws and 2 Hs" protocol is proposed as a way of thinking, with the attempt to clarify Who, Why, When, Where, What, and How to use these treatment options.

Parathyroid Tissue Cryopreservation: Does the Storage Time Affect Viability and Functionality?

Parathyroidectomy is a standard practice to treat recurrent or persistent hyperparathyroidism. However, this can lead to the onset of hypoparathyroidism, treatable with the autotransplantation of parathyroid tissue (PT). Tissue can be transplanted immediately after parathyroidectomy or cryopreserved and transplanted only in case of necessity. Since 2011, the Cord Blood Bank and Cardiovascular Tissue Bank of Emilia-Romagna has been storing PT for potential autologous transplantation. To date, there are highly variable data about the viability and function of PT after thawing. However, it is not clear if the PT quality is affected by different cryopreservation protocols and/or by the storage time. The aim of this study was to assess the ex vivo function and viability of the PTs of ten patients stored in the Bank. Tissue morphology was evaluated before and after cryopreservation through histological investigations. PT function was analyzed by assessing the ability of cryopreserved PT to synthesize and secrete parathyroid hormone (PTH) in response to different calcium concentrations. Moreover, viability and function were also investigated on tissue-isolated cells in culture. These data show that tested tissues appear to be viable and able to produce PTH even after 5 years of storage, and the histological architecture is well preserved.