Phenolic Profiles in Olive Leaves from Different Cultivars in Tuscany and Their Use as a Marker of Varietal and Geographical Origin on a Small Scale.

Olive leaves are a rich source of polyphenols with healthful properties and represent one of the most abundant waste products of olive oil production. The aims of this study were to explore the phenolic composition of olive leaves from the three main Tuscan cultivars (Leccino, Moraiolo and Frantoio) collected in Siena and Grosseto provinces and to investigate the possible use of these compounds as varietal and geographic origin markers. Discriminant factorial analysis (DFA) was used for distinguishing between different cultivars and locations. Apigenin and caffeoyl-secologanoside showed significant differences between cultivars. DFA showed that ligstroside, apigenin and luteolin have the most influence in determining the differences between sites, whereas total polyphenols, olacein and hydroxytyrosol acetate allowed for separation between leaves from the same province. The results of the present study indicate that concentrations of phenolic compounds, measured through high-resolution mass spectrometry, can be used as a marker for both the cultivar and of geographical origin of olive leaves, and possibly of olive-related products, as well as across small geographic scales (less than 50 km distance between sites).

Banking of post-SMILE stromal lenticules for additive keratoplasty: A new challenge for eye banks?

PURPOSE: ReLEx (Refractive Lenticule Extraction) Small Incision Lenticule Extraction (SMILE), the second generation of ReLEx Femtosecond Lenticule Extraction (FLEx), is a minimally invasive, flapless procedure designed to treat refractive errors such as myopia, hyperopia, presbyopia, and astigmatism. This review aims to provide a comprehensive overview of the methods for preserving SMILE-derived lenticules and discusses their potential future applications. METHODS: A narrative literature review was conducted using PubMed, Scopus, and Web of Science databases, focusing on articles published up to January 2024 and available in English. The authors also evaluated the reference lists of the collected papers to identify any additional relevant research. RESULTS: No standardized protocols currently exist for the storage or clinical application of SMILE-derived lenticules. However, these lenticules present a promising resource for therapeutic uses, particularly in addressing the shortage of donor corneal tissues. Their potential applications include inlay and overlay additive keratoplasty, as well as other ocular surface applications. Further research is needed to establish reliable protocols for their preservation and clinical use. CONCLUSION: SMILE-derived lenticules offer significant potential as an alternative to donor corneal tissues. Standardizing their storage and application methods could enhance their use in clinical settings.

Thinking in context: Fibroids-to-uterine volume ratio in pre-surgical fertility evaluation for intramural fibroids.

OBJECTIVE: To explore the utility of the total fibroids-to-uterine volume (FTUV) ratio as a simple, preoperative tool to assist in counseling patients seeking pregnancy who are undergoing myomectomy for intramural (IM) fibroids. STUDY DESIGN: This is an historical cohort study on reproductive-aged patients seeking pregnancy who underwent laparotomic myomectomy for intramural fibroids from January 2017 to December 2021. Only G3 to G5 fibroids, according to the 2011 International Federation of Gynecology and Obstetrics (FIGO) classification, were included. Pre-operative transvaginal ultrasound (TVUS) was performed to measure the volume of intramural myomas (diameter1*diameter2*diameter3*0.52) and to calculate their total volume. The total fibroids-to-uterine volume (FTUV) ratio was calculated as the proportion of the uterine volume occupied by the sum of IM fibroids volumes. RESULTS: A total of 166 women with pre-surgical TVUS evaluation of IM fibroids were included, with a mean age of 36.22 ± 5.15 years. The FTUV ratio was identified as a positive predictor of clinical pregnancy after surgery (adjOR, 1.04; 95 % CI, 1.02-1.06; p = 0.0001), whereas age showed a negative association (adjOR, 0.90; 95 % CI, 0.83-0.98; p = 0.012). Endometrial cavity distortion prior to surgery was also positively associated with pregnancy post-surgery (adjOR, 3.50; 95 % CI, 1.51-8.08; p = 0.003). Consistent results were found for live births, with the FTUV ratio being a significant positive predictor of live birth after surgery (adjOR, 1.03; 95 % CI, 1.01-1.05; p = 0.001) and age showing a negative association (adjOR, 0.88; 95 % CI, 0.80-0.96; p = 0.004). Parity prior to surgery also positively impacted live birth post-surgery (adjOR, 2.65; 95 % CI, 1.30-5.40; p = 0.007). An FTUV ratio threshold of 53.39 % accurately predicted clinical pregnancy in 68.46 % of cases (sensitivity of 71.70 % and specificity of 66.67 %). For live births, a higher FTUV ratio threshold of 59.21 % predicted outcomes accurately in 69.13 % of cases (sensitivity of 65.85 % and specificity of 70.37 %). CONCLUSION: The use of the FTUV ratio in pre-operative ultrasound evaluation of IM fibroids may improve counseling for patients desiring to conceive after myomectomy. By providing a personalized assessment of the amount of myometrial volume occupied by fibroids, the FTUV ratio can help predict fertility outcomes after surgery, enabling better-informed decisions and treatment planning.

Endometriosis and Adenomyosis: Modern Concepts of Their Clinical Outcomes, Treatment, and Management.

Endometriosis and adenomyosis are complex gynecological conditions characterized by diverse clinical presentations, including superficial peritoneal endometriosis (SPE), ovarian endometrioma (OMA), and deep infiltrating endometriosis (DIE). The hallmark features of these pathologies involve the manifestation of pain symptoms and infertility, and approximately 30% of patients are asymptomatic. Despite ongoing research, definitive treatments for these conditions remain elusive, and clinical management primarily revolves around medical or surgical interventions. Recent advancements in our understanding of the efficacy of various treatment modalities, including medical therapy and surgical interventions, have provided clinicians with valuable insights into pain relief and fertility preservation. This review aims to provide an updated overview of the latest literature on clinical outcomes, treatment options, and management strategies for different types of endometriosis. By synthesizing the newest available data, this review seeks to inform clinicians and guide decision making based on factors such as patients' symptom severity, childbearing desire, and overall health.

Mitochondria Transplantation Promotes Corneal Epithelial Wound Healing.

Purpose: The integrity of the corneal epithelium is essential in maintaining normal corneal function. Conditions disrupting the corneal epithelial layer range from chemical burns to dry eye disease and may result in impairment of both corneal transparency and sensation. Identifying factors that regulate corneal wound healing is key for the development of new treatment strategies. Here, we investigated a direct role of mitochondria in corneal wound healing via mitochondria transplantation. Methods: Human corneal epithelial cells (hCECs) were isolated from human corneas and incubated with mitochondria which were isolated from human ARPE-19 cells. We determined the effect of mitochondria transplantation on wound healing and proliferation of hCECs. In vivo, we used a mouse model of corneal chemical injury. Mitochondria were isolated from mouse livers and topically applied to the ocular surface following injury. We evaluated the time of wound repair, corneal re-epithelization, and stromal abnormalities. Results: Mitochondria transplantation induced the proliferation and wound healing of primary hCECs. Further, mitochondria transplantation promoted wound healing in vivo. Specifically, mice receiving mitochondria recovered twice as fast as control mice following corneal injury, presenting both enhanced and improved repair. Corneas treated with mitochondria demonstrated the re-epithelization of the wound area to a multi-layer appearance, compared to thinning and complete loss of the epithelium in control mice. Mitochondria transplantation also prevented the thickening and disorganization of the corneal stromal lamella, restoring normal corneal dehydration. Conclusions: Mitochondria promote corneal re-epithelization and wound healing. Augmentation of mitochondria levels via mitochondria transplantation may serve as an effective treatment for inducing the rapid repair of corneal epithelial defects.

Reproductive outcomes after vaginal repair of isthmocele: A preliminary study and systematic review of the literature.

OBJECTIVE: Although vaginal repair of isthmocele is an effective and safe surgical option, data on reproductive and obstetrical outcomes are lacking. The aim of this study is to evaluate reproductive outcomes of women undergone vaginal repair of isthmocele. We also systematically reviewed the existent literature to offer a general view of available data. STUDY DESIGN: Retrospective analysis of a database prospectively collected between January 2018 and January 2022 at San Raffaele Hospital, Milan, Italy. We included secondary infertile women with ultrasound documented isthmocele who undergone vaginal repair. Post-surgical clinical, reproductive and obstetric outcomes were recorded. An advanced systematic search of the literature up to January 2023 was conducted. RESULTS: 17 women were included. The mean age of the included patients was 37.2 ± 2.7 years. The median of previous caesarian sections was 1 (1-2). One intra-operative complication (5.9 %) was reported (bladder injury, repaired at the time of surgery). At follow up, bleeding was successfully treated in 8 women (8/10; 80 %). Pregnancy was obtained in 7 women (7/17; 41.2 %): the conception was spontaneous in 4 women (4/7; 57.1 %) and trough assisted reproductive technology in 3 patients (3/7; 42.9 %). The mean time from surgery to pregnancy was 10.8 (±6.7) months. One spontaneous abortion was reported (1/7; 14.3 %), while live birth was achieved in 6 pregnancies (6/7; 85.7 %). All deliveries were by caesarian section at a median gestational age of 37.5 (36-38.25) weeks. No obstetrical complications were reported. At the time of caesarean section, no defects on the lower segment were retrieved. Regarding the systematic research, among the 21 studies screened, only 4 articles were included in the review. Pregnancy rate was around 60-70 % with very few obstetrical complications (0.01 %) such as abnormal placentation or preterm birth. CONCLUSIONS: Vaginal repair of isthmocele is a minimally invasive, safe and effective surgical approach in terms of postsurgical residual myometrium tichness. Systematic review to date has found low-quality evidences on the impact of vaginal surgery in the management of secondary infertility and obstetrics outcomes in women with isthmocele.

Shoulder pain management strategies and early functional outcome after arthroscopic rotator cuff tear repair. A randomized controlled study.

BACKGROUND: The management of acute postoperative pain after rotator cuff surgery can be challenging. To our knowledge, there are no data available in the literature correlating satisfactory pain control with improvement in terms of function. The purposes of the present study were to evaluate: 1) pain pattern after arthroscopic rotator cuff repair in patients operated with two different techniques (transosseous vs transosseous equivalent); 2) safety/efficacy of three different pharmacological pain control strategies; 3) possible relationship between a correct shoulder pain management protocol in the early post-operative period and patients' functional improvement. METHODS: 114 patients underwent rotator cuff tear repair, either with a Transosseus or a Transosseus equivalent technique. 62 (54%) were male and 52 (46%) were female. The average age was 59 ± 9 years. They were randomly assigned into three different pain management protocols: Paracetamol as needed (max 3 tablets/day) for 1 week (Protocol A), Paracetamol + Codein 1 tablet three times per day for 7 days (Protocol B), or Paracetamol + Ibuprofen 1 tablet two times per day for 7 days (Protocol C). Immediate passive mobilization of the operated shoulder was allowed. VAS and Passive Flexion values were recorded at 7 (T1), 15 (T2) and 30 (T3) days post-surgery. DASH values were recorded at 90 days post-surgery. All patients were asked to register any kind of signs/symptoms that may appear during drug assumption according to each pain management protocols. RESULTS: All the pain management protocols administered were well tolerated by all the study population, and no adverse signs/symptoms were highlighted during drug assumption. Pain pattern: in both surgical techniques, patients within Protocol A were associated with worst results in terms of mean VAS at each time point examined when compared to Protocol B and C (p < 0,05). In patients within Protocol A, no statistically significant differences were found at each point time examined comparing the two surgical techniques, with the exception of T2, where the TO was associated with an higher VAS value than TOE (p < 0.05). No differences were highlighted in Protocol B and C when comparing the values between two surgical techniques. ROM: in both surgical techniques, patients within Protocol A were associated with worst results in terms of mean PROM at each time point examined when compared to Protocol B and C (p < 0,05). In the TO group, patients within Protocol B had better PROM values at T1 (p < 0,05) and T2 (p < 0,05) compared to Protocol C, but no differences were highlighted at T3. In the TOE group, no statistically significant differences were found between patients within Protocol B and C at each time point examined. DASH: In the TO group, no statistically significant differences were found regarding the DASH values comparing Protocol B vs Protocol C, but they were highlighted comparing the values between Protocol A and Protocol B (p < 0,05), and between Protocol A and Protocol C (p < 0,05). Similar results were recorded in the TOE group. CONCLUSION: Post-operative pain is influenced by the surgical technique used being transosseous more painful in the first 15 days after surgery. Oral anti-inflammatory drugs are a feasible strategy to appropriately control post-operative pain. An association between Paracetamol and either Codein or Ibuprofen can lead to better outcomes in terms of VAS reduction and early recovery of passive ROM.

Assessment of performance and safety of Corneal Chamber hypothermic storage medium and PSS-L corneal rinsing solution in human and porcine corneas.

PURPOSE: To prove the safety and performance of the hypothermic corneal storage medium "Corneal Chamber" and the rinsing solution "PSS-L" in support of the new Conformité Européenne (CE) certification process in accordance with the Medical Device Regulation. METHODS: Fifteen (n=15) human donor corneas and 11 (n=11) porcine corneas were evaluated for the following parameters: endothelial cell density (ECD) and mortality, percentage of hexagonal cells (HEX%), coefficient of cellular area variation (CV%) and corneal transparency at Day 0 and after 14±1 days of storage in Corneal Chamber medium at 2-8°C. Then, the same parameters were assessed after rinsing of corneas in PSS-L for 1 min at room temperature. Evaluation of gentamicin sulfate carryover after corneal storage and PSS-L rinsing was performed by ultra-high performance liquid chromatography analysis on human corneas homogenates. RESULTS: Human and porcine corneas stored in Corneal Chamber medium showed a good overall quality of the tissue according to the quality parameters evaluated. In particular, mean ECD, HEX% and CV% did not show statistically significant changes at the end of storage and endothelial mortality increased to 3.1±3.3 and 7.8±3.5% in human and porcine corneas, respectively. Tissue rinsing with PSS-L did not affect the quality parameters evaluated before and gentamicin sulfate residues were absent in human corneas. CONCLUSIONS: Corneal preservation in Corneal Chamber medium at 2-8°C for 14 days and the corneal rinse with PSS-L are safe and effective procedures allowing the preservation of the corneal quality parameters as well as the complete elimination of gentamicin sulfate from the tissues before transplantation.Cite Now.

Performance outcomes from a DMEK peeling and preparation wet lab.

OBJECTIVE: To evaluate the Descemet membrane endothelial keratoplasty (DMEK) preparation performance of trainee surgeons in an ex vivo human donor cornea DMEK wet lab simulation setting. METHODS: Human donor corneoscleral rims unsuitable for transplantation were obtained from Moorfields Lions Eye Bank. At the wet lab, graft stripping was performed by scoring the peripheral endothelium. The trypan blue positive cells (TBPC) and cell density (cells/mm2-reticule count) were counted manually before and after stripping. The procedural time, peripheral and central tears and complete peel-off were also recorded and analysed. RESULTS: Eight trainee surgeons attended the wet lab each attempting three DMEKs. Between the first and last attempts a significant decrease was seen in the procedural time (17.6 min vs 10.6 min (p<0.05)) and the TBPC % (12.9% vs 3.8% (p<0.05)). The percentage of tears peripherally and centrally also reduced between the first and the last trials (50% vs 13% (p=0.2226) and 38% vs 0% (p=0.1327)). A significant correlation was found between longer peeling times and higher TBPC % (p<0.001) with a 0.7% endothelial mortality increase for each additional minute required to complete the peel. CONCLUSIONS: DMEK wet labs provide a controlled risk-free learning opportunity for trainee surgeons to improve confidence and competence. Wet labs improve the success rate of DMEK graft preparation as well as flatten the learning curve. This emphasises the importance of continued support for the expansion of this valuable learning resource, promoting wider uptake of DMEK surgery.

Quality assurance in corneal transplants: Donor cornea assessment and oversight.

The cornea is the most frequently transplanted human tissue, and corneal transplantation represents the most successful allogeneic transplant worldwide. In order to obtain good surgical outcome and visual rehabilitation and to ensure the safety of the recipient, accurate screening of donors and donor tissues is necessary throughout the process. This mitigates the risks of transmission to the recipient, including infectious diseases and environmental contaminants, and ensures high optical and functional quality of the tissues. The process can be divided into 3 stages: (1) donor evaluation and selection before tissue harvest performed by the retrieval team, (2) tissue analysis during the storage phase conducted by the eye bank technicians after the retrieval, and, (3) tissue quality checks undertaken by the surgeons in the operating room before transplantation. Although process improvements over the years have greatly enhanced safety, quality, and outcome of the corneal transplants, a lack of standardization between centers during certain phases of the process still remains, and may impact on the quality and number of transplanted corneas. Here we detail the donor screening process for the retrieval teams, eye bank operators. and ophthalmic surgeons and examine the limitations associated with each of these stages.

Deep Anterior Lamellar Keratoplasty Using Dehydrated versus Standard Organ Culture-Stored Donor Corneas: Prospective Randomized Trial.

PURPOSE: To compare the outcomes of deep anterior lamellar keratoplasty (DALK) using dehydrated versus standard organ culture-stored donor corneas for eyes with keratoconus. DESIGN: Prospective, randomized, single-center trial conducted in Italy. PARTICIPANTS: Adult patients (age ≥ 18 years) with keratoconus scheduled for elective DALK. METHODS: Patients undergoing successful type 1 bubble pneumatic dissection using a standard DALK technique were randomized during surgery to receive either dehydrated (n = 30) or standard organ culture-stored (n = 30) donor corneas. MAIN OUTCOME MEASURES: The primary study outcome was best spectacle-corrected visual acuity (BSCVA) 12 months after surgery. Secondary outcomes were refractive astigmatism (RA), endothelial cell density (ECD), and complication rates. RESULTS: Postoperative BSCVA did not significantly differ between groups at both time points: mean difference at 6 months was 0.030 logarithm of the minimum angle of resolution (logMAR; 95% confidence interval [CI], -0.53 to 0.10 logMAR; P = 0.471) and at 12 months was -0.013 logMAR (95% CI, -0.10 to 0.08 logMAR; P = 0.764). No significant differences between groups were observed in terms of postoperative RA and ECD at all time points. In the first 3 days after DALK, an epithelial defect was present in 10 patients (33%) in the organ culture cornea group and in 29 patients (97%) in the dehydrated cornea group. Complete re-epithelialization was achieved by day 7 in all patients (100%) in both groups. CONCLUSIONS: The study provides evidence that the use of dehydrated corneas is noninferior to the use of standard organ culture donor corneas for DALK. Corneal tissue dehydration represents a viable solution that can allow long-term cornea preservation and avoid wastage of unused corneas. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Preloaded DMEK with endo-in technique: Standardizing and minimizing the learning curve over 5 years using 599 corneal tissues.

PURPOSE: To report the outcomes of standardizing pre-loaded DMEK with endothelium-inwards and its associated learning curve. METHODS: Between 2017 and 2021, a total of 599 tissues were stripped using 'trephine and strip' method and loaded by folding the tissue as a taco-fold with endothelium-inwards. The folded tissues were pulled inside the funnel of a 2.2 mm IOL cartridge and stored for the desired number of days in organ culture media supplemented with dextran. Donor characteristics, endothelial cell loss (ECL) and mortality assessed by trypan blue positivity before and after stripping, and eventful cases during stripping/loading were recorded. RESULTS: The tissues found unsuitable for transplant after stripping (6.7%) were significantly higher compared with loading (0.67%). Central or peripheral tears, fragility of the tissues, and insufficient endothelial cell density mainly attributed towards the discard rate. Mean ECL from pre-stripping to post-stripping was 0.27% with endothelial cell mortality of 0.64% at the end of stripping. Cumulative endothelial mortality fold change (pre-strip to post-strip) was high in the first two years of operation (18.9%), which reduced to 5.1% in the following three years with significant difference (p = 0.0352). Average tissue wastage (3 operators) from first 1-150 tissues was 3%, which significantly reduced to 0.9% after achieving the learning curve (151-250) (p = 0.0492). CONCLUSION: DMEK graft preparation requires a learning curve. However, an operator with DMEK stripping skills can easily adapt to pre-loading a DMEK graft in endothelium-inwards fashion with minimal learning curve.

Ocular Manifestations in Patients Affected by p63-Associated Disorders: Ectrodactyly-Ectodermal Dysplasia-Clefting (EEC) and Ankyloblepharon-Ectodermal Defects-Cleft Lip Palate (AEC) Syndromes.

BACKGROUND/AIMS: The Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) and Ankyloblepharon-ectodermal defect-cleft lip/palate (AEC) syndromes are rare autosomal dominant diseases caused by heterozygous mutations in the p63 gene. Patients are characterized by abnormalities of the skin, teeth, and hair and have limb defects, orofacial clefting and ectodermal dysplasia. In addition, they often show ocular surface alterations, leading to progressive corneal clouding and eventually blindness. Here, we present 8 cases describing patients affected by EEC (n = 6, with 5 sporadic and 1 familial cases) and AEC (n = 2, both sporadic cases) syndromes. We attempt to provide a description of the ocular disease progression over the years. METHODS: Clinical examinations and monitoring of ocular parameters for the assessment of limbal stem cell deficiency were constantly performed on patients between 2009 and 2023. Quantitative data and comparison with existing cases described in the literature are reported. RESULTS: The therapies supplied to patients were essential for the management of the symptoms, but unfortunately did not halt the progression of the pathology. CONCLUSIONS: A constant monitoring of the patients would help avoid the sudden worsening of symptoms. If the progression of the disease slows down, it would allow for the development of newer therapeutic strategies aimed at correcting the genetic defect.

Identification of the regulatory circuit governing corneal epithelial fate determination and disease.

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.

Preoperative surgeon evaluation of corneal endothelial status: the Viability Control of Human Endothelial Cells before Keratoplasty (V-CHECK) study protocol.

INTRODUCTION: The success of keratoplasty strongly depends on the health status of the transplanted endothelial cells. Donor corneal tissues are routinely screened for endothelial damage before shipment; however, surgical teams have currently no means of assessing the overall viability of corneal endothelium immediately prior to transplantation. The aim of this study is to validate a preoperative method of evaluating the endothelial health of donor corneal tissues, to assess the proportion of tissues deemed suitable for transplantation by the surgeons and to prospectively record the clinical outcomes of a cohort of patients undergoing keratoplasty in relation to preoperatively defined endothelial viability. METHODS AND ANALYSIS: In this multicentre cohort study, consecutive patients undergoing keratoplasty (perforating keratoplasty, Descemet stripping automated endothelial keratoplasty (DSAEK), ultra-thin DSAEK (UT-DSAEK) or Descemet membrane endothelial keratoplasty) will be enrolled and followed-up for 1 year. Before transplantation, the endothelial viability of the donor corneal tissue will be evaluated preoperatively through trypan blue staining and custom image analysis to estimate the overall percentage of trypan blue-positive areas (TBPAs), a proxy of endothelial damage. Functional and structural outcomes at the end of the follow-up will be correlated with preoperatively assessed TBPA values. ETHICS AND DISSEMINATION: The protocol will be reviewed by the ethical committees of participating centres, with the sponsor centre issuing the final definitive approval. The results will be disseminated on ClinicalTrials.gov, at national and international conferences, by partner patient groups and in open access, peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05847387.

P12-A107†Porcine cornea ex vivo model as an alternative to human donor tissues for investigating new preservation conditions.

PURPOSE: Considering the growing shortage of corneal tissues for research, the present study aimed to develop and optimize a porcine cornea model with qualitative features comparable to those of human tissues. METHODS: A new decontamination procedure of porcine eye bulbs was set up and its efficacy as well as endothelial mortality were evaluated. Human corneas unsuitable for transplant and porcine corneas were then compared after storage under hypothermic (4-8°C, Eusol-C, AL.CHI.MI.A. S.R.L) or organ-culture (31-35°C, Tissue-C, AL.CHI.MI.A. S.R.L) storage conditions for 14 days. A new method, based on the semi-automatic analysis of Trypan-blue stained endothelial areas by Fiji software, was developed to quantify the whole endothelium viability. Corneas were assessed for central corneal thickness (CCT), corneal transparency, endothelial morphology, and endothelial cell density (ECD) at days 0, 7, and 14 of storage. Portions of lamellar tissues consisting of Descemet's membrane and endothelial cells were prepared for histological investigations. RESULTS: The new decontamination procedure of porcine eye bulbs resulted in 18% versus 89% ('no decontamination' control) of corneas still contaminated after 28 days of storage at 31°C. The decontamination protocol did not affect endothelium viability, as assessed by the new Fiji-based method. ECD (porcine: 3156 ± 144 cells/mm2; human: 2287 ± 152 cells/mm2), CCT (porcine: 1073 ± 151 µm; human: 581 ± 39 µm), transparency (porcine: 88.6 ± 11.0%; human: 76.3 ± 5.4%), and morphology score (porcine: 4.0 ± 0.0; human: 3.2 ± 0.4) measured in the porcine cornea at day 0 were significantly higher than in human corneas. Nonetheless, the qualitative parameters of porcine and human corneas showed comparable trends during the storage under hypothermic (4-8°C) and organ-culture (31-35°C) conditions for 14 days. CONCLUSION: The presented porcine cornea model represents a reliable and alternative model to human donor tissues for preliminary investigations and can be used for testing new media, substances, drugs, or preservation conditions and their impact on corneal tissue quality and safety. Furthermore, the quantitative method to assess whole endothelium mortality can be implemented at eye banks for the evaluation of corneas intended for transplantation.

P20-A129†An innovative wound healing method reveals a donor and post-mortem time-dependent regeneration of corneal epithelium.

PURPOSE: The aim of this study was to establish and optimize a new and reproducible epithelial wound healing model on human corneas. This assay was used to study the kinetics of epithelial regeneration following a chemical injury. METHODS: Thirty (n=30) human corneas unsuitable for transplant were used for the experiments. Corneas were cultured in Storagix medium (FBOV) at 31°C. Epithelial integrity before the beginning of the experiments (pre-wound) was assessed using the vital dyes trypan blue (TB, TB-S 0.25%, AL.CHI.MI.A. srl) and sodium fluorescein (Fluo). 1-heptanol soaked paper disks (6 mm) were applied in the centre of the corneas for 1' to trigger a chemical damage at the epithelial layer. Afterwards, sodium fluorescein and TB stainings were repeated to quantify the damaged area and to monitor healing progression. The damaged area (mm2) was calculated for each time point with Fiji software. Wound healing rate (HR, mm2/die) was calculated for both Fluo (HRF) and TB (HRTB) measurements using the previously described formula:Arithmetical averages (HRFAVG and HRTBAVG) of HRs were calculated and correlated by Pearson correlation coefficient with the following donor's parameters: age, sex, post-mortem time (PMT, time between death and tissue procurement), stromal defects, septicaemia, body temperature, diabetes. RESULTS: The execution of the heptanol wounding is highly reproducible, as highlighted by Fluo and TB staining. The average time for full recovery from wounding was 3,8 ± 0,41 days for Fluo and 3,5 ± 0,63 days for TB. Fluo and TB stainings are interchangeable as they significantly correlate (Pearson correlation coefficient = 0.630; p>0.05). A negative linear correlation was observed between HR and PMT (HRFAVG: corrected R2: 0.243, p = 0.003; HRTBAVG: corrected R2: 0,132, p = 0.028), but not with the other donors' parameters. CONCLUSION: Our wound/healing model might be of great interest for studies of epithelial regeneration kinetics and validation of drugs for the treatment of ocular defects. The inverse correlation between PMT and HR provides valuable insights for scientists investigating the regenerative properties of the corneal epithelium, as well as for eye bank personnel aiming to preserve the regenerative potential of corneal epithelium.

P16-A111†Factors affecting the density of corneal endothelial cell cultures obtained from donor corneas.

PURPOSE: The shortage of donor corneas represents a worldwide problem, and corneal endothelial cell (CEC) therapy might be a promising alternative approach. CEC can be implanted alone, which has shown limited efficacy, or with a scaffold that holds the cells together as a monolayer tissue, thus imitating Descemet membrane endothelial keratoplasty. We believe that endothelial cell density (ECD) >2000 cells/mm2, a cut-off value that eye banks use to provide quality tissues for transplantation to surgeons, should also be adopted as a parameter to define the quality of CECs as a new Advanced Therapy Medicinal Product for clinical applications in patients with endothelial dystrophies. METHODS: We isolated and cultured CECs from one or more corneas of elderly age donors with ECDs higher than or below 2000 cells/mm2. CEC cultures were carried out on coated plates and on hydrogels with a preformed basement membrane (from TissueGUARD, Germany). Immunofluorescence with antibodies against ZO-1 was performed to evaluate the ECDs of the CEC graft obtained. RESULTS: Our results suggest that primary cultures with ECDs>2000 cells/mm2 can be obtained on coated plated only when (1) CECs are isolated from one or more corneas of young donors; (2) CECs are isolated and pooled together from at least 2 elderly age donor corneas (if ECD>2000 cells/mm2) or 3 elderly age donor corneas (if ECD<2000 cells/mm2). Secondary cultures are all characterized by low ECDs. Hydrogels have been shown to be able to lead to increased ECDs after their release. CONCLUSION: Our protocol highlights the difficulties in obtaining cultures with ECDs>2000 cells/mm2. Despite being achievable with corneas from young donors, this becomes challenging when corneas from elderly donors are used, i.e., the overall majority of those collected by eye banks, particularly when corneas from elderly age donors with ECD<2000 cells/mm2 are considered as a source. One alternative would be to isolate CECs from more corneas, but this might raise the issue of antigenic stimulation, which could eventually lead to transplantation failure. Our strategy to overcome these challenges is the use of a preformed basement membrane as a scaffold for CECs. However, this challenging approach should be investigated more before proceeding to clinical application.

Outcome of rare primary malignant bone sarcoma treated with multimodal therapy: Results from the EUROpean Bone Over 40 Sarcoma Study (EURO-B.O.S.S.).

BACKGROUND: Rare primary malignant bone sarcomas (RPMBS) account for 5%-10% of primary high-grade bone tumors and represent a major treatment challenge. The outcome of patients with RPMBS enrolled in the EUROpean Bone Over 40 Sarcoma Study (EURO-B.O.S.S) is presented. METHODS: Inclusion criteria were as follows: age from 41 to 65 years and a diagnosis of high-grade spindle cell, pleomorphic, or vascular RPMBS. The chemotherapy regimen included doxorubicin 60 mg/m2 , ifosfamide 9 g/m2 , and cisplatin 90 mg/m2 ; postoperative methotrexate 8 g/m2 was added in case of a poor histologic response. Version 2.0 of the Common Terminology Criteria for Adverse Events, Kaplan-Meier curves, log-rank tests, and univariate Cox regression models were used. RESULTS: In total, 113 patients were evaluable for analysis. The median patient age was 52 years (range, 40-66 years), and 67 patients were men. Eighty-eight tumors were categorized as undifferentiated pleomorphic sarcomas (UPS), 20 were categorized as leiomyosarcomas, three were categorized as fibrosarcomas, and two were categorized as angiosarcomas. Eighty-three of 113 tumors were located in the extremities. Ninety-five of 113 patients presented with no evidence of metastases. After a median follow-up of 6.8 years (interquartile range [IQR], 3.5-9.8 years), the 5-year overall survival rate for patients with localized disease was 68.4% (IQR, 56.9%-77.5%), and it was 71.7% (IQR, 58.1%-81.6%) for patients with UPS and 54.9% (IQR, 29.5%-74.5%) for patients with leiomyosarcoma. Grade III-IV hematologic toxicity was reported in 81% patients; 23% had grade II-III neurotoxicity, and 37.5% had grade I-II nephrotoxicity. Five-year overall survival was significantly better for patients with localized disease, for patients who obtained surgical complete remission, and when the primary tumor was located in the extremities. CONCLUSIONS: The survival of patients who had RPMBS in the current series was similar to that of age-matched patients who had high-grade osteosarcoma treated according to the same protocol. An osteosarcoma-like chemotherapy may be proposed in patients who have RPMBS.