Donor Tissue Research Articles

Identifying the greatest surgeon-perceived barriers and challenges in performing corneal transplantation in India.

There is currently no systematic method of assessing limitations in performing corneal transplantation. The purpose of the study was to identify the limitations of performing corneal transplantation in India. Cross-sectional survey study. A total of 148 members of the Cornea Society of India. A survey tool was developed to identify region-specific barriers to performing corneal transplantation. The survey was edited by a marketing firm and reviewed and approved by 4 of the participating corneal surgeons in India. The electronic survey was distributed nationwide to members of the Cornea Society of India between January 1, 2021 and July 1, 2021. Participants were asked to rate the difficulty or ease of 11 predetermined steps/variables of the corneal transplantation process using a Likert scale. One hundred forty-eight practicing cornea surgeons participated in the survey (21% response rate). The most common barrier was difficulty with finding donors who fulfilled the screening criteria for transplantation (55%). The second most common barrier was patient/family willingness to donate organs (32%). The third most common barrier was the inability to store tissue after it was prepared by the eye bank (20%). The two variables least commonly identified as barriers were operating room staff availability and using the donor tissue before expiration. The greatest barrier to corneal transplantation in India was finding donors who fulfilled screening criteria. Additional studies into the contributing factors are underway to inform governing bodies on resource allocation to enhance procurement of quality donor tissues.

Digital light processing 3D bioprinting of biomimetic corneal stroma equivalent using gelatin methacryloyl and oxidized carboxymethylcellulose interpenetrating network hydrogel.

Corneal blindness, a leading cause of visual impairment globally, has created a pressing need for alternatives to corneal transplantation due to the severe shortage of donor tissues. In this study, we present a novel interpenetrating network hydrogel composed of gelatin methacryloyl (GelMA) and oxidized carboxymethyl cellulose (OxiCMC) for bioprinting a biomimetic corneal stroma equivalent. We tested different combinations of GelMA and OxiCMC to optimize printability and subsequently evaluated these combinations using rheological studies for gelation and other physical, chemical, and biological properties. Using digital light processing (DLP) bioprinting, with tartrazine as a photoabsorber, we successfully biofabricated three-dimensional constructs with improved shape fidelity, high resolution, and excellent reproducibility. The bioprinted constructs mimic the native corneal stroma's curvature, with central and peripheral thicknesses of 478.9 ± 56.5 µm and 864.0 ± 79.3 µm, respectively. The dual crosslinking strategy, which combines Schiff base reaction and photocrosslinking, showed an improved compressive modulus (106.3 ± 7.7 kPa) that closely matched that of native tissues (115.3 ± 13.6 kPa), without relying on synthetic polymers, toxic crosslinkers, or nanoparticles. Importantly, the optical transparency of tartrazine-containing corneal constructs was comparable to the native cornea following phosphate-buffered saline washing. Morphological analyses using scanning electron microscopy confirmed the improved porosity, interconnected network, and structural integrity of the GelMA-OxiCMC hydrogel, facilitating better nutrient diffusion and cell viability. In vitro biological assays demonstrated high cell viability (>93%) and desirable proliferation of human corneal keratocytes within the biofabricated constructs. Our findings indicate that the GelMA-OxiCMC hydrogel system for DLP bioprinting presents a promising alternative for corneal tissue engineering, offering a potential solution to the donor cornea shortage and advancing regenerative medicine for corneal repair.&#xD.

Corneal donation and its relationship with public awareness

Only one donor tissue is available for seventy patients awaiting a corneal graft, indicating a global scarcity of corneal graft tissue. The lack of awareness and information among potential donors and their families is directly linked to this deficiency in eye donation practices. There is a much greater need for corneal tissue than there are eye donations. Prompt corneal procurement demonstrates its maximum utility. A maximum of six hours from the time of death is the optimal window for corneal procurement.

4.5-Year Follow-up with a Novel Corneal Endothelial Prosthesis.

Refractory corneal edema is the foremost reason for endothelial corneal transplantation (EK) in the world. Descemet membrane endothelial keratoplasty (DMEK) and Descemet stripping automated endothelial keratoplasty (DSAEK) offer good clinical outcomes. However, human donor tissue is limited in availability and has a complex logistical chain. Many patients in need do not have access to EK. Success of the human donor tissue depends on its quality, the number of rebubbles, repeat keratoplasties, and patient-related risk factors. The use of an endothelial corneal keratoprosthesis could benefit patients with a high risk for graft failure and human graft rejection, and patients in underserved areas where tissue banking is unavailable. Case cohort series of 10 eyes of 10 patients implanted with the EndoArt, with a follow-up of up to 4.5 years. Outcome measures are device adherence, central corneal thickness, visual acuity, and incidence of adverse events. In the first seven eyes, the EndoArt was implanted in eyes with low visual potential within the first-in-human (FIH) trial, and in the three remaining eyes, implantation was performed because of either guarded posterior or anterior segment prognosis or the patient's wishes. In 9/10 eyes, device adherence was obtained and maintained throughout the follow-up period. Mean corneal thickness decreased from 927 + SD 241 microns to 621 + SD 140 microns. In 6/10 eyes, visual acuity improved from a mean preoperative value of logMAR 1.94 + 0.63 to a mean postoperative value of logMAR 0.8 + 0.64. No pathological thinning or corneal metabolic changes were seen. The surgical technique was developed during the follow-up period. The EndoArt provided a stable decrease in central corneal thickness, improved vision in 60% of eyes, and had no device-related adverse events. The EndoArt is an additional treatment modality for special cases like repeat corneal graft failure in the population of the Western world, or as a primary procedure in underserved areas worldwide.

Immunology in corneal transplantation-From homeostasis to graft rejection.

Immunology depends on maintaining a delicate balance within the human body, and disruptions can result in conditions such as autoimmune diseases, immunodeficiencies, and hypersensitivity reactions. This balance is especially crucial in transplantation immunology, where one of the primary challenges is preventing graft rejection. Such rejection can lead to organ failure, increased patient mortality, and higher healthcare costs due to the limited availability of donor tissues relative to patient needs. Xenotransplantation, like using porcine corneas for human transplants, offers a potential solution to the donor tissue shortage but faces substantial immunological rejection issues. To prevent rejection in both allo- and xenotransplantation, a deep understanding of how the body maintains immunological balance is essential, particularly since achieving tolerance to non-self tissues is considered the "holy grail" of the field. The cornea, the most frequently transplanted solid organ, has a high acceptance rate due to its immune-privileged status and serves as an ideal model for studying graft rejection mechanisms that disrupt tolerance. However, multiple immune pathways complicate our understanding of these mechanisms. This review examines the rejection mechanisms in corneal transplantation, identifying key cells involved and potential therapeutic strategies to induce and maintain immunological tolerance in both allo- and xenografts across various transplants.

Evaluation of the Palatal Features in Relation to Graft Harvesting in the Saudi Population.

Background and Objectives: The palate's morphological characteristics are of great importance, especially in periodontology, where the palatine tissue represents a source of tissue graft for multiple mucogingival surgeries. This study aimed to estimate the amount of donor tissue available through the average palatal height and average location of the greater palatine artery in the Saudi population according to age and gender. Materials and Methods: Digital casts for adult Saudi patients at the age of 18-60 years old with a mean age of 37.76 ± 12.68 years were collected and analyzed using EXOCAD software. The digital casts were evaluated, and measurements of arch width at molars and canines, palatal vault height (PVH), palatal height index (PHI), and extension of the palatal rugae were registered. Finally, the amount available for soft tissue graft harvesting was calculated. Results: Overall, 109 maxillary casts for Saudi patients, 52 (47.7%) males and 57 (52.3%) females, were analyzed. The maxillary inter-molar arch width, inter-canine width, and palatal vault height significantly differed between males and females (p < 0.05). The mean PHI was 45.51% ± 8.12%, and 27.5% were classified as orthostaphyline, while 72.5% were categorized as hypsistaphyline, with no significant difference between the genders. The mean maximum graft width was 11.45 mm, and the graft width was significantly different between males and females (p < 0.005), while the mean maximum graft length was 17.78 mm, and the graft length showed no significant difference. Conclusions: The results of this study provide specific clinical guidelines for periodontal procedures by emphasizing the importance of gender-specific anatomical considerations. Data on graft dimensions and palatal measurements will enable the exact planning of soft tissue harvesting to minimize surgical risks and optimize mucogingival surgery outcomes in the Saudi population.

Estimation of melanocytic count in different donor areas in non-segmental vitiligo before and after narrow band ultraviolet B phototherapy

Background The gluteal region and thigh are common donor sites used during surgical treatment of stable nonsegmental vitiligo (NSV). The outcome of transplantation could be improved if the donor site has higher melanocyte density. Aim To compare the count of melanocytes in the gluteal region and thigh, and evaluate the impact of narrow band ultraviolet B (NB-UVB) phototherapy on the quantity of melanocytes in both areas. Methods In this study, 20 stable NSV patients and five controls were included. Before and after 18 sessions of NB-UVB, two 3 mm punch biopsies were obtained from the normal skin of the thigh and gluteal region. Melan A was used for immunohistochemically labeling, and a count of melanocytes was performed on each sample. Results Before NB-UVB therapy, there was no significant difference in the number of melanocytic cells in the gluteal region and thigh in NSV patients (P=0.777) or between cases and controls. In NSV cases, the melanocytic count increased significantly in the thigh and gluteal region (P=0.002 for both), with no difference observed between the two sites (P=0.430). In the gluteal region, there was a significant positive correlation between the total cumulative joules (P=0.025, r=0.511) and the percentage change of melanocytic count and skin phototype (P=0.001, r=0.677). Conclusion In NSV patients, the melanocytic count in the normal skin of the thigh and gluteal region is similar. Following NB-UVB, this count significantly increased in both sites similarly. Both sites are equally appopriate for harvesting donor tissue for surgery in NSV cases preferably after NB-UVB phototherapy.

Random Perforator Flap : Some Experiences with Keystone Flap

Background: In reconstructing challenging defects, surgeons are considered fairly fortunate when they are able to obtain a similar donor tissue quality to that of the missing tissue; in regards to their color, texture, size, and the ease of donor transfer to the defect. Several methods may be used, which frequently include the free tissue transfers using microvascular anastomoses bearing their specific consequences. We report a select of challenging cases which were successfully reconstructed using the Keystone flaps and avoid microvascular anastomoses, where otherwise the free tissue transfers would be the typical option for closure in such defects. Patient and Method: Nine cases of relatively large defect in various locations were reconstructed using the Keystone flaps supplied by either non-identified perforators or identified reliable perforators. Result: Out of the 9 defects located on various region of the body (lumbar, thorax, dorsum of the foot, plantar of the foot, posterior leg, sacrum, and cervicofacial) only the first 2 cases had identifiable perforators. All flaps survived completely without problem of vascularization. Summary: The Keystone flap is a useful and reliable random perforator-based flap even when the perforator vessels are not identified.

Elevation of Granulocyte Colony Stimulating Factor in Human AMD Donor RPE-Choroid.

Choroidal inflammation, complement deposition, and accumulation of C-reactive protein (CRP) are involved in age-related macular degeneration (AMD) pathology. The pro-inflammatory signals that regulate immune cell recruitment in the choroid of patients with AMD remain to be determined. We performed cytokine profiling of human AMD and age-matched control donor tissue to identify inflammatory molecules upregulated in AMD tissue. Protein was isolated from 25 AMD and 21 control donor RPE/choroid macular punches. Total protein was quantified, and 50 µg assayed for expression of 40 cytokines using an inflammation array. We validated the elevated expression of granulocyte colony stimulating factor (G-CSF) protein by ELISA in a second cohort of 22 control and 26 AMD donors. To identify an AMD associated stressor responsible for upregulating G-CSF we assayed for changes in G-CSF protein secretion in RPE/choroid organ cultures treated with the monomeric (m)CRP, an inflammatory protein elevated in AMD. Using a multiplex array, we identified elevated G-CSF protein in the choroid of AMD donors compared to age-matched non-AMD controls. Differential expression of G-CSF was confirmed via ELISA in an independent cohort of samples (P = 0.01). The mCRP, which is deposited in AMD choroids, increased G-CSF protein secretion in RPE/choroid organ cultures. Single nuclei RNA sequencing identified choroidal endothelial cells and fibroblasts as the primary cell types responsible for increased G-CSF secretion in response to mCRP. The G-CSF receptor is expressed primarily by choroidal macrophages and dendritic cells and anti-G-CSFR colocalizes with anti-CD45 and anti-CD68 in human donor choroid tissue. Elevated G-CSF expression in AMD donor tissue as a result of increased levels of mCRP may be involved in immune cell recruitment in AMD contributing to inflammatory stress in the choroid.

The Chondrogenic Potential of the Bovine Tendon Sheath - a Novel Source of Stem Cells for Cartilage Repair.

The human hand is traumatized more frequently than any other bodily part. Trauma and pathological processes (e.g., rheumatoid arthritis, osteoarthritis) commonly implicate the finger joints and specifically damage also the layer of articular cartilage. Endeavors are now being made to surgically repair such cartilage lesions biologically using tissue-engineering approaches that draw on donor cells and/or donor tissues. The tendon sheaths, particularly their inner layers, i.e., the peritendineum, surround the numerous tendons in the hand. The peritendineum is composed of mesenchymal tissue. We hypothesize that this tissue harbors pluripotent mesenchymal stem cells and thus could be used for cartilage repair, irrespective of the donor's age. Using a bovine model (young calves vs. adult cows), the pluripotentiality of the peritendineal stem cells, namely, their osteogenicity, chondrogenicity, and adipogenicity, was investigated by implementing conventional techniques. Subsequently, the chondrogenic potential of the peritendineal tissue itself was analyzed. Its differentiation into cartilage was induced by the application of specific growth factors (members of the TGF-β-superfamily). The characteristics of the tissue formed were evaluated structurally (immuno) histochemically, histomorphometrically, and biochemically (gene expression and protein level). Our data confirm that the bovine peritendineum contains stem cells whose pluripotentiality is independent of donor age. This tissue could also be induced to differentiate into cartilage, likewise, irrespective of the donor's age. Preliminary investigations with adult human peritendineal biopsy material derived from the hand's peritendineal flexor tendon sheaths revealed that this tissue can also be induced to differentiate into cartilage.

3D-Printed Polymer Scaffolds for Vascularized Bone Regeneration Using Mineral and Extracellular Matrix Deposition

Abstract Purpose Trauma, injury, disease, infection, congenital deformities, and non-union after a fracture can lead to significant loss of bone tissue resulting in large bone defects. If left untreated, this can lead to decreased bone strength, stability, and function as well as long-term malformations. We present a novel, pre-vascularized 3D-printed biodegradable scaffold mimicking the architecture of native bone as a bone graft alternative to promote vascularized bone regeneration. Methods Scaffolds with a highly porous central trabecular section surrounded by an outer cortical section modeled after the bone’s osteons were 3D printed in polylactic acid (PLA). Hydroxyapatite (HA) posts were incorporated to improve mechanical strength. A soak-freeze technique was used to introduce additional porosity to support the recruitment, proliferation, and differentiation of stem cells. Scaffolds were mineralized to provide cues for osteoconduction and osteoinduction. They were also pre-vascularized to promote the differentiation of stem cells along the vascular lineage. Results Compression mechanical testing showed the addition of HA posts improved mechanical strength. Using the soak-freeze technique, micropores in the range of 0–10 µm were introduced. Osteogenic differentiation capability of the scaffolds was verified in vitro through the estimation of osteocalcin (OC) produced by the cells seeded on them and by staining for alkaline phosphatase. Differentiation of stem cells along the vascular lineage within the scaffold was confirmed via the estimation of vascular endothelial growth factor (VEGF-A) and by staining for CD31, a marker for vascular differentiation. Conclusion This novel scaffold incorporated with cues necessary to promote the regeneration of bone and its vasculature shows promise as an alternative to currently used bone grafts. Lay Summary Significant bone loss caused by trauma, infection, or disease results in large defects that are currently treated using bone grafts—autografts (taken from the same patient), allografts and xenografts (donor tissue), or synthetic grafts. We have developed a tissue-engineered alternative that mimics the architecture of natural bone and has cues to promote both the regeneration of bone and its vasculature. These are fabricated using 3D printing (3DP) technology, providing cost-effective, customizable alternatives to conventional bone grafts.

Tissue Processing and Isolation of Primary Fibroblasts from the Human Vagina.

Pelvic organ prolapse is a disorder that seriously impacts the quality of life of women. It occurs when muscles and ligaments weaken and cause pelvic organs to drop lower in the pelvis, creating a bulge in the vagina.Surgery to correct pelvic organ prolapse has been a mainstay treatment. Recently, there has been growing interest in studying the tissue composition of patients with prolapse at the cellular level. There is currently little consensus regarding the effect of donor or patient age on cell-based therapies. Current published protocols for vaginal fibroblast isolation either concentrate on premenopausal tissue or neglect to comment on the age of donor tissue. Most existing protocols use animal models. The consistency of human vaginal tissue is denser than the tissues used in most protocols. In this study, human vaginal tissue was obtained primarily from older donors, which likely contributed to the failure of existing protocols. The aim of this study is to describe a standard protocol for reliably acquiring human vaginal fibroblasts, regardless of donor age and menopausal status. Results were reproduced using tissue from nine separate donors who underwent pelvic organ prolapse surgery. Six patients were postmenopausal, with the oldest donor being 78 years old. The median age of the tissue donors was 59. Here, we describe a reliable method for generating a fibroblast-enriched single-cell suspension using a combination of enzymatic and mechanical dissociation and cell suspension pooling of multiple vaginal biopsies from a single donor. Reliable isolation of human vaginal primary fibroblasts may be useful in the study of pelvic organ prolapse as well as microbiome-host interactions.

Comparison of clinical outcomes, complications and patient satisfaction following deep anterior lamellar keratoplasty and penetrating keratoplasty

BackgroundKeratoplasty is a surgical procedure in which a damaged or diseased cornea is replaced with healthy donor tissue, thereby restoring vision. Recent advancements have led to the replacement of penetrating keratoplasty (PKP) with the more selective deep anterior lamellar keratoplasty (DALK) procedure, especially for treating keratoconus. Although DALK typically has a shorter recovery time, less pain and a lower risk of rejection, PKP is still being performed for more severe corneal diseases. A comparative study of clinical profiles, treatment outcomes and patient-reported satisfaction will provide valuable insights into the cost-effectiveness, impact on quality of life and ability of each procedure to treat different pathologies of the cornea. Here, we aimed to compare the clinical and subjective outcomes of DALK with those of the PKP at a single center in the Kingdom of Saudi Arabia.MethodsThis retrospective cohort study included eyes that underwent either PKP or DALK from January 2017 to January 2021. The demographic features, indications, best corrected visual acuity (BCVA) and complications of the patients were recorded for both groups, analyzed and compared. A subgroup of eyes with keratoconus was analyzed separately and compared to the larger group. A 6-item survey was conducted via telephone to assess patient satisfaction and expectation, and the results were compared between the two procedures. The chi-square test or Fisher’s exact test for categorical variables or the t test or Kruskal‒Wallis test for continuous variables were used as appropriate for all comparisons, and the level of significance was set at α = 0.05.ResultsA total of 97 patients were included. PKP and DALK were performed on 63 and 39 eyes, respectively. Patients who underwent DALK were younger (mean ± standard deviation 31 ± 10.82 years versus 43 ± 26.89 years for patients who underwent PKP). The most frequent indication for PKP was keratoconus (35.5%); however, in 97.4% of the eyes undergoing DALK, the indication was keratoconus. In both groups, visual acuity and refractive error improved, but the postoperative corrected distance visual acuity in the DALK group (0.3 log MAR) was noticeably greater than that in the PKP group (0.6 log MAR). Compared with PKP, DALK may carry a lower risk of early graft edema and rejection. Overall, the reported postoperative patient satisfaction was similar for both procedures.ConclusionThe findings highlight the effectiveness of PKP and DALK in improving visual acuity and emphasize the importance of considering patient-reported outcomes in evaluating success. DALK has been demonstrated to be beneficial for protecting the corneal endothelium and lowering the risk of complications and graft rejection.

Harnessing the Power of MicroRNA Cargoes in Small Extracellular Vesicles Released from Fresh-Frozen Human Brain Sections.

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic acids (microRNA, mRNA, DNA). The microRNA sEV cargo has potential utility as a powerful non-invasive disease biomarker due to sEV's ability to traverse biological barriers (e.g., blood-brain barrier) and become accessible through various body fluids. Despite numerous studies on sEV biomarkers in body fluids, identifying tissue or cell-specific sEV subpopulations remains challenging, particularly from the brain. Our study addresses this challenge by adapting existing methods to isolate sEVs from minimal amounts of frozen human brain sections using size exclusion chromatography (SEC). After ethical approval, approximately 250 µg of fresh-frozen human brain tissue (obtained from Manchester Brain Bank [UK]) was sliced from the 3 donor tissues and incubated in collagenase type 3/Hibernate-E solution, with intermediate agitation, followed by serial centrifugation and filtration steps. Then, sEVs were isolated using the SEC method and characterized by following MISEV guidelines. Before isolating RNA from within these sEVs, the solution was treated with Proteinase-K and RNase-A to remove any non-sEV extracellular RNA. The RNA quantity and quality were checked and processed further for qPCR and small RNA sequencing experiments. The presence of sEVs was confirmed through fluorescence nanoparticle tracking analysis (fNTA) and western blot for surface markers (CD9, CD63, CD81). Size distribution (50-200 nm) was confirmed by NTA and electron microscopy. The total RNA concentration within lysed sEVs ranged from 3-9 ng/µL and was used for successful quantification by qPCR for selected candidate microRNAs. Small RNA sequencing on MiSeq provided high-quality data (Q >32) with 1.4-5 million reads per sample. This method enables efficient isolation and characterization of sEVs from minimal brain tissue volumes, facilitating non-invasive biomarker research and holds promise for equitable disease biomarker studies, offering insights into neurodegenerative diseases and potentially other disorders.

Optimizing Myelomeningocele Repair: Assessing the Role of Synthetic Acellular Dermal Regeneration Templates in Complex Closures.

Myelomeningoceles threaten newborns with central nervous system infectious risk. While some myelomeningoceles can be repaired fetally, limited donor tissue in newborns makes covering a substantial defect challenging. This study evaluated the effectiveness of acellular dermal regeneration templates (ADRT) in safely healing refractory myelomeningoceles. Seven myelomeningocele repair cases using ADRT (Integra LifeSciences, Plainsboro, NJ) at an academic children's hospital from April 2020 to June 2023 were reviewed. Patients had unsuccessful closure attempts through fetoscopic, postnatal, or revision surgeries by neurosurgery and plastic surgery, leading to complications that required ADRT to protect the dural repair and promote quicker granulation. The case series included 3 male and 4 female patients, with a median delivery age of 37 weeks (IQR: 33-37). Three underwent fetoscopic repairs, and 4 had postnatal repairs within 48 hours of birth. Six patients required ADRT placement due to failed primary repair. One patient failed fetoscopic closure and required immediate ADRT placement following an emergent cesarean delivery. The median wound size covered was 12cm2 (range, 4-20cm2), and the median hospital stay was 84 days (IQR: 43-105). Three weeks post-ADRT placement, 4 patients showed healthy granulation tissue, and the external silicone layer was removed. Three patients needed additional ADRT for complete wound coverage and successful granulation. After granulation, all wounds eventually epithelialized by secondary intention, with no postoperative infection or wound dehiscence observed. ADRT can aid in wound healing and protect dural repair in myelomeningoceles, offering a viable option for complex or failed primary closures with limited donor tissue.

Orthobiologic Products: Preservation Options for Orthopedic Research and Clinical Applications.

The biological products used in orthopedics include musculoskeletal allografts-such as bones, tendons, ligaments, and cartilage-as well as biological therapies. Musculoskeletal allografts support the body's healing process by utilizing preserved and sterilized donor tissue. These allografts are becoming increasingly common in surgical practice, allowing patients to avoid more invasive procedures and the risks associated with donor site morbidity. Bone grafting is one of the most frequently used procedures in orthopedics and traumatology. Biologic approaches aim to improve clinical outcomes by enhancing the body's natural healing capacity and reducing inflammation. They serve as an alternative to surgical interventions. While preliminary results from animal studies and small-scale clinical trials have been promising, the field of biologics still lacks robust clinical evidence supporting their efficacy. Biological therapies include PRP (platelet-rich plasma), mesenchymal stem cells (MSCs)/stromal cells/progenitor cells, bone marrow stem/stromal cells (BMSCs), adipose stem/stromal cells/progenitor cells (ASCs), cord blood (CB), and extracellular vesicles (EVs), including exosomes. The proper preservation and storage of these cellular therapies are essential for future use. Preservation techniques include cryopreservation, vitrification, lyophilization, and the use of cryoprotective agents (CPAs). The most commonly used CPA is DMSO (dimethyl sulfoxide). The highest success rates and post-thaw viability have been achieved by preserving PRP with a rate-controlled freezer using 6% DMSO and storing other cellular treatments using a rate-controlled freezer with 5% or 10% DMSO as the CPA. Extracellular vesicles (EVs) have shown the best results when lyophilized with 50 mM or 4% trehalose to prevent aggregation and stored at room temperature.

Crescentic Autograft-Graft Salvage Technique for Undersized Donor Lenticules in Superficial Anterior Lamellar Keratoplasty.

To describe the use of crescentic autograft as a graft salvage technique for inadvertently undersized donor lenticules in superficial anterior lamellar keratoplasty (SALK). A prospective interventional study included 5 eyes of 4 patients who underwent SALK and had an inadvertently undersized donor lenticule. The described technique involves the preparation of a crescent-shaped graft from the host lenticule itself that is matched to fit into the remaining bare recipient stromal bed. At 6-week follow-up, the crescentic autograft was uniformly epithelized along with the donor tissue. There were no incidences of graft dehiscence, rejection, or infection. Crescentic autograft from the host lenticule offers a simple, easy-to perform, and unique technique to manage cases of undersized donor lenticules in SALK with no additional risks.

Donor Tissue Preservation Time Impact on Keratolimbal Allograft Outcomes.

This study sought to determine the effect of donor preservation time and other donor tissue factors on the clinical outcomes of keratolimbal allograft (KLAL) transplantation. In this retrospective chart review of patients who underwent KLAL transplantation, electronic health records were reviewed for relevant patient ocular and surgical history. Donor tissue factors were collected from donor reports. The primary outcome measure was time to epithelialization. Exclusion criteria included prior ocular surface stem cell transplantation, loss to follow-up, missing data from medical records, indication for surgery other than limbal stem cell deficiency, and early removal of KLAL tissue. Regression analysis was performed to compare time to epithelialization and donor tissue factors. Subgroup analysis was performed using the Student t test to evaluate the impact of donor preservation time on time to epithelialization. A total of 154 eyes were identified of which 122 were included for analysis. The mean time to epithelialization was 55 days and median was 35 days. No donor tissue factors were found to affect total time to corneal epithelialization. Subgroup analysis comparing the effect of different total preservation times on time to epithelialization did not reveal any statistically significant associations. No relationship was found between time to donor tissue preservation and time to epithelialization after KLAL surgery. It appears that limbal stem cells can be safely stored for up to 9 days in corneal storage solution in hypothermic temperatures without clinical consequences.

Dissecting the cell microenvironment of ovarian endometrioma through single-cell RNA sequencing.

Ovarian endometrioma (OE), also known as "chocolate cysts," is a cystic mass that develops in the ovaries due to endometriosis and is a common gynecological condition characterized by the growth of endometrial tissue outside the uterus, leading to symptoms such as dysmenorrhea, pelvic pain, and infertility. However, the precise molecular and cellular mechanisms driving this pathophysiology remain largely unknown, posing challenges for diagnosis and treatment. Here, we employed integrated single-cell transcriptomic profiling of over 52,000 individual cells from endometrial tissues of OE patients and healthy donors and identified twelve major cell populations. We identified notable alterations in cell type-specific proportions and molecular signatures associated with OE. Notably, the activation of IGFBP5+ macrophages with pro-inflammatory properties, NK cell exhaustion, and aberrant proliferation of IQCG+ and KLF2+ epithelium are key features and may be the potential mechanisms underlying the pathogenesis of OE. Collectively, our data contribute to a better understanding of OE at the single cell level and may pave the way for the development of novel therapeutic strategies.

Free fillet flap: Outcomes in emergency microsurgical reconstruction of upper and lower limb

INTRODUCTIONDespite its infrequency, “spare-part surgery” represents a reconstructive strategy that uses segments of amputated limbs as donor tissue to preserve function in other injured anatomical regions. The purpose was to review our 14-year experience in emergency microsurgical reconstruction of traumatised limbs using free fillet flaps obtained from non-salvageable amputated parts. MATERIALS AND METHODSA retrospective observational study was conducted on patients who underwent urgent free fillet flap transfer for upper or lower limb reconstruction between 2007 and 2021. The characteristics and levels of injury, as well as the flap designs, functional outcomes and complications were documented. RESULTSA series of eight clinical cases is presented. One osteo-tendino-cutaneous and seven fasciocutaneous flaps were filleted from non-replantable amputated limbs for free transfer. Stable coverage of the dorsal aspect of the hand and fingers was achieved in three patients, and limb shortening was avoided in five patients. An open fracture of the thumb with bone defect, three below-knee stumps and one trans-metatarsal level were preserved. The mean Q-DASH score for upper limb reconstructions was 16.75 points. In all cases of lower limb reconstruction, the nerves were repaired, resulting in a protective sensitivity (S3 level) with no pressure ulcer development during the follow-up period. All patients were able to return to normal prosthetic ambulation with a mean Amputee Mobility Predictor Pro score of 45 points. Two haematomas, one surgical wound dehiscence, and two immediate revisions of venous anastomoses were registered. CONCLUSIONSThe microvascular filleted flap demonstrated versatility in the emergency reconstruction of complex limb injuries, providing stable coverage and optimised limb length preservation within a single major surgical procedure while eliminating morbidity from any other uninjured donor site.