Dynamic tracking of human umbilical cord mesenchymal stem cells (hUC-MSCs) following intravenous administration in mice model.

Introduction: In the past decades, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have sparked interest in cellular therapy due to their immunomodulatory properties. Nevertheless, the fate of hUC-MSCs in the body remains poorly understood. This study aimed to investigate the biodistribution, homing and clearance of systemically administered hUC-MSCs in healthy BALB/c mice model. Methods: hUC-MSCs were labelled with GFP-Luc2 protein, followed by characterisation with flow cytometry. Upon intravenous infusion of transduced hUC-MSCs into the healthy BALB/c mice, the cells were dynamically monitored through the bioluminescent imaging (BLI) approach. Results: Transduction of hUC-MSCs with GFP-Luc2 not only preserved the characteristics of MSCs, but also allowed live monitoring of transduced cells in the mice model. Upon systemic administration, BLI showed that transduced hUC-MSCs first localised predominantly in the lungs of healthy BALB/c mice and mainly remained in the lungs for up to 3 days before eventually cleared from the body. At terminal sacrifice, plasma chemistry biomarkers remained unchanged except for C-peptide levels, which were significantly reduced in the hUC-MSCs group. Histopathological findings further revealed that hUC-MSCs infusion did not cause any adverse effects and toxicity to lung, liver and heart tissues. Conclusions: Collectively, systemically administrated hUC-MSCs was safe and demonstrated dynamic homing capacity before eventually disappearing from the body.

Preclinical assessments of safety and tumorigenicity of very high doses of allogeneic human umbilical cord mesenchymal stem cells.

Human umbilical cord-mesenchymal stem cells (hUC-MSCs) have been widely investigated as a new therapeutic agent to treat injuries and inflammatory-mediated and autoimmune diseases. Previous studies have reported on the safety of low-dose infusion of hUC-MSCs, but information on the cell behaviour at higher doses and frequency of injection of the cells remains uncertain. The aim of the present study was to demonstrate the safety and efficacy of hUC-MSCs by Cytopeutics® (Selangor, Malaysia) from low to an extremely high dose in different monitoring periods in healthy BALB/c mice as well as assessing the tumorigenicity of the cells in B-NDG SCID immunocompromised mice. Umbilical cord from two healthy human newborns was obtained and the isolation of the hUC-MSCs was performed based on previous established method. Assessment of the cells at different doses of single or multiple administrations was performed on healthy BALB/c mice in dose range finding, sub-acute (7 d and 28 d) and sub-chronic periods (90 d). Tumorigenicity potential of Cytopeutics® hUC-MSCs was also evaluated on B-NDG immunocompromised mice for 26 wk. Single or multiple administrations of Cytopeutics® hUC-MSCs up to 40 × 106 cells per kilogramme of body weight (kg BW) were found to have no adverse effect in terms of clinical symptoms, haematology and other laboratory parameters, and histology examination in healthy BALB/c mice. hUC-MSCs were also found to reduce pro-inflammatory cytokines (IL-6 and TNF-α) in a dose-dependent manner. No sign of tumor formation was observed in B-NDG mice in the 26-wk tumorigenicity assessment. Single or multiple administration of allogenic Cytopeutics® hUC-MSCs was safe even at very high doses, is non-tumorigenic and did not cause adverse effects in mice throughout the evaluation periods. In addition, Cytopeutics® hUC-MSCs exhibited immunomodulatory effect in a dose-dependent manner.

Headway and the remaining hurdles of mesenchymal stem cells therapy for bronchopulmonary dysplasia.

OBJECTIVE: Preterm infants are at a high risk of developing BPD. Although progression in neonatal care has improved, BPD still causes significant morbidity and mortality, which can be attributed to the limited therapeutic choices for BPD. This review discusses the potential of MSC in treating BPD as well as their hurdles and possible solutions. DATA SOURCES: The search for data was not limited to any sites but was mostly performed on all clinical trials available in ClinicalTrials.gov as well as on PubMed by applying the following keywords: lung injury, preterm, inflammation, neonatal, bronchopulmonary dysplasia and mesenchymal stem cells. STUDY SELECTIONS: The articles chosen for this review were collectively determined to be relevant and appropriate in discussing MSC not only as a potential treatment strategy for curbing the incidence of BPD but also including insights on problems regarding MSC treatment for BPD. RESULTS: Clinical trials regarding the use of MSC for BPD had good results but also illustrated insights on problems to be addressed in the future regarding the treatment strategy. Despite that, the clinical trials had mostly favourable reviews. CONCLUSION: With BPD existing as a constant threat and there being no permanent solutions, the idea of regenerative medicine such as MSC may prove to be a breakthrough strategy when it comes to treating BPD. The success in clinical trials led to the formulation of prospective MSC-derived products such as PNEUMOSTEM®, and there is the possibility of a stem cell medication and permanent treatment for BPD in the near future.

Mesenchymal Stem Cell-Derived Extracellular Vesicles: Progress and Remaining Hurdles in Developing Regulatory Compliant Quality Control Assays.

Regenerative medicine is shaping into a new paradigm and could be the future medicine driven by the therapeutic capabilities shown by mesenchymal stem cell-derived extracellular vesicles (MSC-EVs). Despite the advantages and promises, the therapeutic effectiveness of MSC-EVs in some clinical applications is restricted due to inconsistent manufacturing process and the lack of stringent quality control (QC) measurement. In particular, QC assays which are crucial to confirm the safety, efficacy, and quality of MSC-EVs available for end use are poorly designed. Hence, in this review, characterization of MSC-EVs and quality control guidelines for biologics are presented, with special attention given to the description of technical know-how in developing QC assays for MSC-EVs adhering to regulatory guidelines. The remaining challenges surrounding the development of potency and stability of QC assays are also addressed.

An Affordable Approach of Mesenchymal Stem Cell Therapy in Treating Perianal Fistula Treatment.

The application of stem cells to treat perianal fistula due to Crohn's disease has attracted a lot of interest in recent decades. Though still a popular procedure, the existing surgical methods may be an ideal form of therapy since the recurrence rate is high, which affects the quality of life badly. Stem cell therapy offers to be a better solution in treating PF, but the utilisation is often restricted because of the manufacturing cost. Hence in this review, the selection of suitable cell sources, the use of bioreactors and preconditioning MSCs as well as modified stem cells will be discussed for a more affordable as compared with the current MSC therapy towards PF. We anticipate that exploring these approaches may give a complete picture in understanding stem cells in order to make them effective and affordable for long-term therapeutic applications.

Genetic Modification as a New Approach to Ameliorate the Therapeutic Efficacy of Stem Cells in Diabetic Retinopathy.

Over the last decades, the strategy of using stem cells has gained a lot of attention in treating many diseases. Recently, DR was identified as one of the common complications experienced by diabetic patients around the world. The current treatment strategy needs to be addressed since the active progression of DR may lead to permanent blindness. Interestingly, varieties of stem cells have emerged to optimize the therapeutic effects. It is also known that stem cells possess multilineage properties and are capable of differentiating, expanding in vitro and undergoing genetic modification. Moreover, modified stem cells have shown to be an ideal resource to prevent the degenerative disease and exhibit promising effects in conferring the migratory, anti-apoptotic, anti-inflammatory and provide better homing for cells into the damaged tissue or organ as well promoting healing properties. Therefore, the understanding of the functional properties of the stem cells may provide the comprehensive guidance to understand the manipulation of stem cells making them useful for long-term therapeutic applications. Hence in this review the potential use and current challenges of genetically modified stem cells to treat DR will be discussed along with its future perspectives.

Corrigendum: Transplanted Erythropoietin-Expressing Mesenchymal Stem Cells Promote Pro-survival Gene Expression and Protect Photoreceptors From Sodium Iodate-Induced Cytotoxicity in a Retinal Degeneration Model.

[This corrects the article DOI: 10.3389/fcell.2021.652017.].

The potential role of mesenchymal stem cells in modulating antiageing process.

Ageing and age-related diseases share some basic origin that largely converges on inflammation. Precisely, it boils down to a common pathway characterised by the appearance of a fair amount of proinflammatory cytokines known as inflammageing. Among the proposed treatment for antiageing, MSCs gained attention in recent years. Since mesenchymal stem cells (MSCs) can differentiate itself into a myriad of terminal cells, previously it was believed that these cells migrate to the site of injury and perform their therapeutic effect. However, with the more recent discovery of huge amounts of paracrine factors secreted by MSCs, it is now widely accepted that these cells do not engraft upon transplantation but rather unveil their benefits through excretion of bioactive molecules namely those involved in inflammatory and immunomodulatory activities. Conversely, the true function of these paracrine changes has not been thoroughly investigated all these years. Hence, this review will describe in detail on ways MSCs may capitalize its paracrine properties in modulating antiageing process. Through a comprehensive literature search various elements in the antiageing process, we aim to provide a novel treatment perspective of MSCs in antiageing related clinical conditions.

Transplanted Erythropoietin-Expressing Mesenchymal Stem Cells Promote Pro-survival Gene Expression and Protect Photoreceptors From Sodium Iodate-Induced Cytotoxicity in a Retinal Degeneration Model.

Mesenchymal stem cells (MSC) are highly regarded as a potential treatment for retinal degenerative disorders like retinitis pigmentosa and age-related macular degeneration. However, donor cell heterogeneity and inconsistent protocols for transplantation have led to varied outcomes in clinical trials. We previously showed that genetically-modifying MSCs to express erythropoietin (MSCEPO) improved its regenerative capabilities in vitro. Hence, in this study, we sought to prove its potential in vivo by transplanting MSCsEPO in a rat retinal degeneration model and analyzing its retinal transcriptome using RNA-Seq. Firstly, MSCsEPO were cultured and expanded before being intravitreally transplanted into the sodium iodate-induced model. After the procedure, electroretinography (ERG) was performed bi-weekly for 30 days. Histological analyses were performed after the ERG assessment. The retina was then harvested for RNA extraction. After mRNA-enrichment and library preparation, paired-end RNA-Seq was performed. Salmon and DESeq2 were used to process the output files. The generated dataset was then analyzed using over-representation (ORA), functional enrichment (GSEA), and pathway topology analysis tools (SPIA) to identify enrichment of key pathways in the experimental groups. The results showed that the MSCEPO-treated group had detectable ERG waves (P <0.05), which were indicative of successful phototransduction. The stem cells were also successfully detected by immunohistochemistry 30 days after intravitreal transplantation. An initial over-representation analysis revealed a snapshot of immune-related pathways in all the groups but was mainly overexpressed in the MSC group. A subsequent GSEA and SPIA analysis later revealed enrichment in a large number of biological processes including phototransduction, regeneration, and cell death (Padj <0.05). Based on these pathways, a set of pro-survival gene expressions were extracted and tabulated. This study provided an in-depth transcriptomic analysis on the MSCEPO-treated retinal degeneration model as well as a profile of pro-survival genes that can be used as candidates for further genetic enhancement studies on stem cells.

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles ameliorate airway inflammation in a rat model of chronic obstructive pulmonary disease (COPD).

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an incurable and debilitating chronic disease characterized by progressive airflow limitation associated with abnormal levels of tissue inflammation. Therefore, stem cell-based approaches to tackle the condition are currently a focus of regenerative therapies for COPD. Extracellular vesicles (EVs) released by all cell types are crucially involved in paracrine, extracellular communication. Recent advances in the field suggest that stem cell-derived EVs possess a therapeutic potential which is comparable to the cells of their origin. METHODS: In this study, we assessed the potential anti-inflammatory effects of human umbilical cord mesenchymal stem cell (hUC-MSC)-derived EVs in a rat model of COPD. EVs were isolated from hUC-MSCs and characterized by the transmission electron microscope, western blotting, and nanoparticle tracking analysis. As a model of COPD, male Sprague-Dawley rats were exposed to cigarette smoke for up to 12 weeks, followed by transplantation of hUC-MSCs or application of hUC-MSC-derived EVs. Lung tissue was subjected to histological analysis using haematoxylin and eosin staining, Alcian blue-periodic acid-Schiff (AB-PAS) staining, and immunofluorescence staining. Gene expression in the lung tissue was assessed using microarray analysis. Statistical analyses were performed using GraphPad Prism 7 version 7.0 (GraphPad Software, USA). Student's t test was used to compare between 2 groups. Comparison among more than 2 groups was done using one-way analysis of variance (ANOVA). Data presented as median ± standard deviation (SD). RESULTS: Both transplantation of hUC-MSCs and application of EVs resulted in a reduction of peribronchial and perivascular inflammation, alveolar septal thickening associated with mononuclear inflammation, and a decreased number of goblet cells. Moreover, hUC-MSCs and EVs ameliorated the loss of alveolar septa in the emphysematous lung of COPD rats and reduced the levels of NF-κB subunit p65 in the tissue. Subsequent microarray analysis revealed that both hUC-MSCs and EVs significantly regulate multiple pathways known to be associated with COPD. CONCLUSIONS: In conclusion, we show that hUC-MSC-derived EVs effectively ameliorate by COPD-induced inflammation. Thus, EVs could serve as a new cell-free-based therapy for the treatment of COPD.

Hypoxic-Mediated Oxidative Stress Condition and Hydroxyapatite-Inducing Osteogenic Differentiation of Human Mesenchymal Stem Cells: A Mathematical Modeling Study.

Avascular necrosis (AVN) of the bones remains a major clinical challenge. Fractures in the talus, the scaphoid, and the neck of the femur are especially challenging to heal due to the low blood vessel network and the lack of collateral blood supply. These fractures are associated with high rates of nonunion and increased infections that require repeated operations. Conventional treatments by autografting or allografting bone replacement and synthetic bone implants have limitations, including the invasiveness of operative procedures, tissue supply insufficiency, and the risk of host rejection. The advancement in tissue engineering has revealed the potential of stem cells as restorative agents for bone injuries. The administration of mesenchymal stem cells (MSCs) into the talus, the scaphoid, and the neck of the femur could produce enhanced osteogenesis via the manipulation of MSC culture conditions. In this study, we used hydroxyapatite as the nanomaterial, and hypoxic milieu to enhance MSC differentiation capacity into the osteogenic lineage, allowing for more rapid and efficient bone cell replacement treatment. Our results demonstrate 1% oxygen and 12.5 µg/mL of hydroxyapatite (HAP) as the optimal conditions to incorporate the osteogenic medium for the osteogenic induction of MSCs. We also established a proof of concept that the addition of HAP and hypoxic conditions could augment the osteoinductive capacity of MSCs. We also developed an accurate mathematical model to support future bone cell replacement therapy.

High Dose of Intravenous Allogeneic Umbilical Cord-Derived Mesenchymal Stem Cells (CLV-100) Infusion Displays Better Immunomodulatory Effect among Healthy Volunteers: A Phase 1 Clinical Study.

BACKGROUND: Mesenchymal stem cells (MSCs) express growth factors and other cytokines that stimulate repair and control the immune response. MSCs are also immunoprivileged with low risk of rejection. Umbilical cord-derived MSCs (UCMSCs) are particularly attractive as an off-the-shelf allogeneic treatment in emergency medical conditions. We aim to determine the safety and efficacy of intravenous allogeneic infusion of UCMSCs (CLV-100) by Cytopeutics® (Selangor, Malaysia) in healthy volunteers, and to determine the effective dose at which an immunomodulatory effect is observed. Methodology. Umbilical cord samples were collected after delivery of full-term, healthy babies with written consent from both parents. All 3 generations (newborn, parents, and grandparents) were screened for genetic mutations, infections, cancers, and other inherited diseases. Samples were transferred to a certified Good Manufacturing Practice laboratory for processing. Subjects were infused with either low dose (LD, 65 million cells) or high dose (HD, 130 million cells) of CLV-100 and followed up for 6 months. We measured cytokines using ELISA including anti-inflammatory cytokines interleukin 1 receptor antagonist (IL-1RA), interleukin 10 (IL-10), pro-/anti-inflammatory cytokine interleukin 6 (IL-6), and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). RESULTS: 11 healthy subjects (LD, n = 5; HD, n = 6; mean age of 55 ± 13 years) were recruited. All subjects tolerated the CLV-100 infusion well with no adverse reaction throughout the study especially in vital parameters and routine blood tests. At 6 months, the HD group had significantly higher levels of anti-inflammatory markers IL1-RA (705 ± 160 vs. 306 ± 36 pg/mL; p = 0.02) and IL-10 (321 ± 27 vs. 251 ± 28 pg/mL; p = 0.02); and lower levels of proinflammatory marker TNF-α (74 ± 23 vs. 115 ± 15 pg/mL; p = 0.04) compared to LD group. CONCLUSION: Allogeneic UCMSCs CLV-100 infusion is safe and well-tolerated in low and high doses. Anti-inflammatory effect is observed with a high-dose infusion.

Looking into dental pulp stem cells in the therapy of photoreceptors and retinal degenerative disorders.

Blindness and vision impairment are caused by irremediable retinal degeneration in affected individuals worldwide. Cell therapy for a retinal replacement can potentially rescue their vision, specifically for those who lost the light sensing photoreceptors in the eye. As such, well-characterized retinal cells are required for the replacement purposes. Stem cell-based therapy in photoreceptor and retinal pigment epithelium transplantation is well received, however, the drawbacks of retinal transplantation is the limited clinical protocols development, insufficient number of transplanted cells for recovery, the selection of potential stem cell sources that can be differentiated into the target cells, and the ability of cells to migrate to the host tissue. Dental pulp stem cells (DPSC) belong to a subset of mesenchymal stem cells, and are recently being studied due to its high capability of differentiating into cells of the neuronal lineage. In this review, we look into the potential uses of DPSC in treating retinal degeneration, and also the current data supporting its application.

Dental pulp stem cells therapy overcome photoreceptor cell death and protects the retina in a rat model of sodium iodate-induced retinal degeneration.

Blindness and vision loss contribute to irreversible retinal degeneration, and cellular therapy for retinal cell replacement has the potential to treat individuals who have lost light sensitive photoreceptors in the retina. Retinal cells are well characterized in function, and are a subject of interest in cellular replacement therapy of photoreceptors and the retinal pigment epithelium. However, retinal cell transplantation is limited by various factors, including the choice of potential stem cell source that can show variability in plasticity as well as host tissue integration. Dental pulp is one such source that contains an abundance of stem cells. In this study we used dental pulp-derived mesenchymal stem cells (DPSCs) to mitigate sodium iodate (NaIO3) insult in a rat model of retinal degeneration. Sprague-Dawley rats were first given an intravitreal injection of 3 × 105 DPSCs as well as a single systemic administration of NaIO3 (40 mg/kg). Electroretinography (ERG) was performed for the next two months and was followed-up by histological analysis. The ERG recordings showed protection of DPSC-treated retinas within 4 weeks, which was statistically significant (* P ≤ .05) compared to the control. Retinal thickness of the control was also found to be thinner (*** P ≤ .001). The DPSCs were found integrated in the photoreceptor layer through immunohistochemical staining. Our findings showed that DPSCs have the potential to moderate retinal degeneration. In conclusion, DPSCs are a potential source of stem cells in the field of eye stem cell therapy due to its protective effects against retinal degeneration.

Retinal degeneration rat model: A study on the structural and functional changes in the retina following injection of sodium iodate.

Retinal disorders account for a large proportion of ocular disorders that can lead to visual impairment or blindness, and yet our limited knowledge in the pathogenesis and choice of appropriate animal models for new treatment modalities may contribute to ineffective therapies. Although genetic in vivo models are favored, the variable expressivity and penetrance of these heterogeneous disorders can cause difficulties in assessing potential treatments against retinal degeneration. Hence, an attractive alternative is to develop a chemically-induced model that is both cost-friendly and standardizable. Sodium iodate is an oxidative chemical that is used to simulate late stage retinitis pigmentosa and age-related macular degeneration. In this study, retinal degeneration was induced through systemic administration of sodium iodate (NaIO3) at varying doses up to 80 mg/kg in Sprague-Dawley rats. An analysis on the visual response of the rats by electroretinography (ERG) showed a decrease in photoreceptor function with NaIO3 administration at a dose of 40 mg/kg or greater. The results correlated with the TUNEL assay, which revealed signs of DNA damage throughout the retina. Histomorphological analysis also revealed extensive structural lesions throughout the outer retina and parts of the inner retina. Our results provided a detailed view of NaIO3-induced retinal degeneration, and showed that the administration of 40 mg/kg NaIO3 was sufficient to generate disturbances in retinal function. The pathological findings in this model reveal a degenerating retina, and can be further utilized to develop effective therapies for RPE, photoreceptor, and bipolar cell regeneration.

Micro-Computed Tomography Detection of Gold Nanoparticle-Labelled Mesenchymal Stem Cells in the Rat Subretinal Layer.

Mesenchymal stem cells are widely used in many pre-clinical and clinical settings. Despite advances in molecular technology; the migration and homing activities of these cells in in vivo systems are not well understood. Labelling mesenchymal stem cells with gold nanoparticles has no cytotoxic effect and may offer suitable indications for stem cell tracking. Here, we report a simple protocol to label mesenchymal stem cells using 80 nm gold nanoparticles. Once the cells and particles were incubated together for 24 h, the labelled products were injected into the rat subretinal layer. Micro-computed tomography was then conducted on the 15th and 30th day post-injection to track the movement of these cells, as visualized by an area of hyperdensity from the coronal section images of the rat head. In addition, we confirmed the cellular uptake of the gold nanoparticles by the mesenchymal stem cells using transmission electron microscopy. As opposed to other methods, the current protocol provides a simple, less labour-intensive and more efficient labelling mechanism for real-time cell tracking. Finally, we discuss the potential manipulations of gold nanoparticles in stem cells for cell replacement and cancer therapy in ocular disorders or diseases.

Relocation of cryopreserved umbilical cord blood samples using a high-capacity dry shipper to a new laboratory: a cord blood banking experience.

BACKGROUND: Processed umbilical cord blood (UCB) must be stored at cryogenic temperature at all times to maintain the quality and viability of the cells. However, a challenge is presented in the form of moving a large number of cryopreserved UCB samples to a new location. In this report, we share our experience on relocating more than 100,000 units of cryopreserved UCB samples stored in 12 liquid nitrogen freezers (LNFs) to our new laboratory. STUDY DESIGN AND METHODS: For quality control purposes, 2 weeks before relocation, donor UCB samples were processed, cryopreserved, and stored in each LNF. On relocation day, half of the samples were retrieved to determine total nucleated cell count, percentage of CD34+ cells, and cell viability as controls for later comparison. UCB samples were transferred into dry shippers before being relocated to the new laboratory. Upon arrival, LNFs were serviced before transferring UCB samples back into its original location within the LNF. The remaining donor UCB samples were retrieved and analyzed for the same tests mentioned. RESULTS: We found no significant differences in pre- and postrelocation values of the tests performed. CONCLUSION: All UCB samples were successfully relocated into the new laboratory without affecting the quality.

Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane.

The cornea can be damaged by a variety of clinical disorders or chemical, mechanical, and thermal injuries. The objectives of this study were to induce bone marrow mesenchymal stem cells (BMSCs) to corneal lineage, to form a tissue engineered corneal substitute (TEC) using BMSCs, and to treat corneal surface defects in a limbal stem cell deficiency model. BMSCs were induced to corneal lineage using limbal medium for 10 days. Induced BMSCs demonstrated upregulation of corneal stem cell markers; ß1-integrin, C/EBPδ, ABCG2, and p63, increased protein expression of CK3 and p63 significantly compared with the uninduced ones. For TEC formation, passage 1 BMSCs were trypsinized and seeded on amniotic membrane in a transwell co-culture system and were grown in limbal medium. Limbal stem cell deficiency models were induced by alkaline injury, and the TEC was implanted for 8 weeks. Serial slit lamp evaluation revealed remarkable improvement in corneal regeneration in terms of corneal clarity and reduced vascularization. Histologic and optical coherence tomography analyses demonstrated comparable corneal thickness and achieved stratified epithelium with a compact stromal layer resembling that of normal cornea. CK3 and p63 were expressed in the newly regenerated cornea. In conclusion, BMSCs can be induced into corneal epithelial lineage, and these cells are viable for the formation of TEC, to be used for the reconstruction of the corneal surface in the limbal stem cell deficient model.

Spontaneous bilateral corneal perforation in stevens- johnsons syndrome-a challenge in management.

A 42-year-old man from Ghana presented with bilateral painful corneal perforations following ingestion of a sulphur-based antibiotic. Emergency bilateral penetrating keratoplasty was performed, with restoration of globe integrity. However, surgical complications arose such as non-healing epithelial defect, secondary infection, graft dehiscence, and mounting intraocular pressure. This case illustrates the challenges faced in managing corneal grafts in patients with already compromised ocular surfaces.

Suturing techniques and postoperative management in penetrating keratoplasty in the United Kingdom.

AIMS: To report on the suturing techniques and aspects of postoperative management in penetrating keratoplasty in the United Kingdom. METHODS: A postal questionnaire was sent to 137 ophthalmic consultants identified from a Royal College of Ophthalmology database as having a special interest in anterior segment surgery. The questionnaire surveyed surgeon preferences for surgical and suturing technique for penetrating keratoplasty surgery, and the postoperative care of corneal grafts. RESULTS: In all, 68% of questionnaires were completed and returned: 73% of respondents used a Flieringa ring or equivalent, 94% routinely used cardinal sutures, with 50.5% removing them at the end of the procedure. The most common suturing technique for routine penetrating keratoplasty was a single continuous suture (35%). In these cases, a 10/0 nylon suture was used by 89%. Sixty-six percent changed their technique in high-risk cases, 52% used a 3-1-1 knot, and 75% made a distinction between a reef and granny knot, with 76% using a reef. Thirty percent buried the knots within the donor material, and 29% within the host tissue. Twenty-five percent had no routine time for graft suture removal, but 41% removed them between 1 and 2 years post-surgery. After suture removal, 98% used steroids and 88% used topical antibiotics. Thirty-four percent stopped topical steroids before suture removal, with 38% stopping topical steroids more than 3 months prior to suture removal. CONCLUSION: This survey demonstrates that there is considerable variation in suturing techniques and postoperative care for penetrating keratoplasty. These significant variations in practice need to be considered when interpreting outcomes and research.