Manufacturing of human corneal endothelial grafts.

PURPOSE: Human corneal endothelial cells (HCECs) play a significant role in maintaining visual function. However, these cells are notorious for their limited proliferative capacity in vivo. Current treatment of corneal endothelial dysfunction resorts to corneal transplantation. Herein we describe an ex vivo engineering method to manufacture HCEC grafts suitable for transplantation through reprogramming into neural crest progenitors. METHODS: HCECs were isolated by collagenase A from stripped Descemet membrane of cadaveric corneoscleral rims, and induced reprogramming via knockdown with p120 and Kaiso siRNAs on collagen IV-coated atelocollagen. Engineered HCEC grafts were released after assessing their identity, potency, viability, purity and sterility. Phase contrast was used for monitoring cell shape, graft size, and cell density. Immunostaining was used to determine the normal HCEC phenotype with expression of N-cadherin, ZO-1, ATPase, acetyl-α-tubulin, γ-tubulin, p75NTR, α-catenin, ß-catenin, and F-actin. Stability of manufactured HCEC graft was evaluated after transit and storage for up to 3 weeks. The pump function of HCEC grafts was measured by lactate efflux. RESULTS: One HCEC graft suitable for corneal transplantation was generated from 1/8th of the donor corneoscleral rim with normal hexagonal cell shape, density, and phenotype. The manufactured grafts were stable for up to 3 weeks at 37 °C or up to 1 week at 22 °C in MESCM medium and after transcontinental shipping at room temperature by retaining normal morphology (hexagonal, >2000 cells/mm2, >8 mm diameter), phenotype, and pump function. CONCLUSIONS: This regenerative strategy through knockdown with p120 and Kaiso siRNAs can be used to manufacture HCEC grafts with normal phenotype, morphology and pump function following prolonged storage and shipping.

Fornix deepening reconstruction in conjunctivochalasis surgery.

PURPOSE: To assess the extent of inferior fornix shortening in conjunctivochalasis (CCh) and to evaluate whether fornix deepening reconstruction can restore the fornix tear reservoir in patients with CCh. MATERIALS AND METHODS: This was a retrospective review of five patients (3 unilateral and 2 bilateral eyes, total 7 eyes) with CCh who underwent fornix deepening reconstruction with conjunctival recession and amniotic membrane transplantation. Postsurgical outcome measures included changes in fornix depth with correlation to basal tear volumes, symptoms, corneal staining, and conjunctival inflammation. RESULTS: For the three patients with unilateral surgery, both the fornix depth (8.3 ± 1.5 mm) and wetting length (9.3 ± 8.5 mm) of the operative eyes were less than the fellow eyes (10.3 ± 1.5 mm and 10.3 ± 8.5 mm, respectively). At 5.3 ± 2.7 months (range 1.7-8.7) postoperatively, the fornix depth increased significantly by 2.0 ± 1.1 mm (P = 0.02). Deepening of the fornix depth was accompanied by overwhelming symptomatic relief (91.5%) that could be subdivided into complete relief (87.5%) and partial relief (4%) of symptoms, with blurred vision being the most notably relieved symptom (P = 0.03). Furthermore, superficial punctate keratitis and conjunctival inflammation were significantly improved at follow-up (P = 0.008 and 0.05, respectively). CONCLUSION: Deepening of the fornix to restore the tear reservoir is an important surgical objective that may change the tear hydrodynamic state to provide a stable tear film and improve outcomes in CCh.

Suppression of TGF-ß1 signaling by Matrigel via FAK signaling in cultured human trabecular meshwork cells.

The trabecular meshwork (TM) is composed of TM cells and beams of the extracellular matrix, together contributing to aqueous humor (AH) outflow resistance. Herein, we validated that our culture system on 2D Matrigel expressed putative TM markers and myocilin, of which the latter was upregulated by dexamethasone. Continuous passage of these cells on 2D Matrigel resulted in a gradual loss of expression of these markers. However, such a loss was restored by seeding cells in 3D Matrigel where expression of TM markers was further upregulated upon continuous passage. In contrast, TM cells seeded on fibronectin, collagen I/IV, or laminin lost expression of these markers and turned into myofibroblasts with expression of αSMA, which were dose-dependently upregulated by TGF-ß1/TGF-ß2. TM cells in 3D Matrigel also expressed TGF-ß1/TGF-ß3 despite challenge of TGF-ß1. The maintenance of TM phenotype by 3D Matrigel was linked to inhibition of canonical TGF-ß signaling and activation of pFAK-pSrc-pP190RhoGAP-P120RasGAP signaling. These findings indicate that basement membrane matrix with low rigidity plays an active role in maintaining TM phenotype in the presence of TGF-ß1 and shed light on its physiological role. Furthermore, abnormal matrices may perpetuate the pathological TM phenotype when the level of TGF-ß2 is elevated in glaucoma patients.

HC-HA/PTX3 from amniotic membrane reverts senescent limbal niche cells to Pax6+ neural crest progenitors to support limbal epithelial progenitors.

Quiescence and self-renewal of human corneal epithelial progenitor/stem cells (LEPC) are regulated by the limbal niche, presumably through close interaction with limbal (stromal) niche cells (LNC). Paired box homeotic gene 6 (Pax6), a conserved transcription factor essential for eye development, is essential for proper differentiation of limbal and corneal epithelial stem cells. Pax6 haploinsufficiency causes limbal stem cell deficiency, which leads to subsequent corneal blindness. We previously reported that serial passage of nuclear Pax6+ LNC resulted in the gradual loss of nuclear Pax6+ and neural crest progenitor status, the latter of which was reverted upon recovery of Pax6. These findings suggest Pax6 plays a pivotal role in supporting the self-renewal of LEPC in limbal niche. Herein, we show that HC-HA/PTX3, a unique matrix purified from amniotic membrane (AM) and consists of heavy chain 1of inter-α-trypsin inhibitor covalently linked to hyaluronic acid and complexed with pentraxin 3, is capable of reverting senescent LNC to nuclear Pax6+ neural crest progenitors that support self-renewal of LEPC. Such reversion is causally linked to early cell aggregation mediated by activation of C-X-C chemokine receptor type 4 (CXCR4)-mediated signaling followed by activation of bone morphogenetic protein (BMP) signaling. Furthermore, CXCR4-mediated signaling, but not BMP signaling, controls recovery of the nuclear Pax6+ neural crest progenitors. These findings not only explain why AM helps in vivo and ex vivo expansion of human LEPC, but they also illuminate the potential role of HC-HA/PTX3 as a surrogate matrix niche that complements stem cell-based therapies in regenerative medicine.

Advances in Descemet membrane endothelial keratoplasty / 中华实验眼科杂志

With relatively low rejection rate and better visual prognosis, Descemet membrane endothelial keratoplasty (DMEK) has become the mainstream surgery for the treatment of endothelial dysfunction in some developed countries, but it has not been applied widely in China due to technical difficulties, the long learning curve, shallow anterior chamber of Chinese people, and the fact that domestic corneal endothelial lesions are often accompanied with other complex eye diseases.In this review, the indications, donor graft preparation including donor selection, graft preparation techniques and visualization of graft, key surgical techniques including the implantation, unwrapping and positioning of graft, postoperative complications including graft detachment, high intraocular pressure, rejection, endothelial cell loss, graft survival rate, and visual prognosis of DMEK were reviewed.

Niche regulation of limbal epithelial stem cells: HC-HA/PTX3 as surrogate matrix niche.

Homeostasis of the corneal epithelium is ultimately maintained by stem cells that reside in a specialized microenvironment within the corneal limbus termed palisades of Vogt. This limbal niche nourishes, protects, and regulates quiescence, self-renewal, and fate decision of limbal epithelial stem/progenitor cells (LEPCs) toward corneal epithelial differentiation. This review focuses on our current understanding of the mechanism by which limbal (stromal) niche cells (LNCs) regulate the aforementioned functions of LEPCs. Based on our discovery and characterization of a unique extracellular matrix termed HC-HA/PTX3 (Heavy chain (HC1)-hyaluronan (HA)/pentraxin 3 (PTX3) complex, "-" denotes covalent linkage; "/" denotes non-covalent binding) in the birth tissue, i.e., amniotic membrane and umbilical cord, we put forth a new paradigm that HC-HA/PTX3 serves as a surrogate matrix niche by maintaining the in vivo nuclear Pax6+ neural crest progenitor phenotype to support quiescence and self-renewal but prevent corneal fate decision of LEPCs. This new paradigm helps explain how limbal stem cell deficiency (LSCD) develops in aniridia due to Pax6-haplotype deficiency and further explains why transplantation of HC-HA/PTX3-containing amniotic membrane prevents LSCD in acute chemical burns and Stevens Johnson syndrome, augments the success of autologous LEPCs transplantation in patients suffering from partial or total LSCD, and assists ex vivo expansion (engineering) of a graft containing LEPCs. We thus envisage that this new paradigm based on regenerative matrix HC-HA/PTX3 as a surrogate niche can set a new standard for regenerative medicine in and beyond ophthalmology.

Novel Gyrovirus genomes recovered from free-living pigeons in Southern Brazil.

Wild birds carry a number of infectious agents, some of which may have pathogenic potential for the host and others species, including humans. Domestic pigeons (Columba livia) are important targets of study since these increasingly cohabit urban spaces, being possible spillover sources of pathogens to humans. In the present study, two genomes (PiGyV_Tq/RS/Br and PiGyV_RG/RS/Br), representative of Gyrovirus genus, family Anelloviridae, were detected in sera of free-living pigeons collected in Southern Brazil. The genomes exhibit less than 50% identity to previously described members of Gyrovirus genus, suggesting that they constitute a new viral species circulating in pigeons, to which the name "pigeon gyrovirus (PiGyV)" is proposed. The current study characterizes these two PiGyV genomes which, to date, are the first gyrovirus species identified in domestic pigeons.

Self-Retained Cryopreserved Amniotic Membrane for the Management of Corneal Ulcers.

PURPOSE: To evaluate the clinical outcomes of self-retained cryopreserved amniotic membrane (cAM) for the treatment of corneal ulcers. METHODS: This was a single-center, retrospective review of consecutive patients with non-healing corneal ulcers that underwent treatment with self-retained cAM (PROKERA® Slim). The primary outcome measure was time to complete corneal epithelialization. Ocular discomfort, corneal staining, corneal signs, and visual acuity were assessed at 1 week, 1 month, 3 months, and 6 months. Complications, adverse events, and ulcer recurrence were also recorded. RESULTS: A total of 13 eyes (13 patients) with recalcitrant corneal ulcers were included for analysis, 9 (69%) of which progressed from neurotrophic keratitis (NK). Prior to cAM application, patients used conventional treatments such as artificial tears (n = 11), antibiotics (n = 11), ointment (n = 11), steroids (n = 6), and antivirals (n = 3). Self-retained cAMs (n = 1.5 ± 0.8) were placed for 6.8 ± 3.4 days, during which time antibiotics were continued. Four cases (31%) were subsequently treated with bandage contact lens (n = 3) and tarsorrhaphy (n = 1). All corneal ulcers healed in a median of 14 days (range: 4-43). This was accompanied by a significant improvement in ocular discomfort, corneal staining, and corneal signs at 1 week, 1 month, 3 months, and 6 months (P<.05). Recurrence was noted in one case. No adverse events were observed. CONCLUSION: Self-retained cAM may be a valuable, in-office treatment option for healing recalcitrant corneal ulcers of various etiologies, especially those with underlying NK. Further prospective, controlled studies are warranted.

Comment on: "A novel approach to peatlands as archives of total cumulative spatial pollution loads from atmospheric deposition of airborne elements complementary to EMEP data: Priority pollutants (Pb, Cd, Hg)" by Ewa Miszczak, Sebastian Stefaniak, Adam Michczynski, Eiliv Steinnes and Irena Twardowska.

A recent paper by Miszczak et al. (2020) examines metal contamination of mires in Poland and Norway. The authors conclude that lead (Pb) records in ombrotrophic peatlands cannot be used to reconstruct the chronological history of anthropogenic activities due to post-depositional mobility of the metal. We contest this general conclusion which stands in contrast with a significant body of literature demonstrating that Pb is largely immobile in the vast majority of ombrotrophic peatlands. Our aim is to reaffirm the crucial contribution that peat records have made to our knowledge of atmospheric Pb contamination. In addition, we reiterate the necessity of following established protocols to produce reliable records of anthropogenic Pb contamination in environmental archives.

HC-HA/PTX3 Purified From Human Amniotic Membrane Reverts Human Corneal Fibroblasts and Myofibroblasts to Keratocytes by Activating BMP Signaling.

Purpose: Fibrosis or scarring is a pathological outcome of wound healing and is characterized by terminally differentiated myofibroblasts. Heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) is a unique matrix component purified from amniotic membrane that exerts an anti-inflammatory effect. Herein, we investigate whether HC-HA/PTX3 can also exert an antiscarring effect. Methods: Human corneal fibroblasts and myofibroblasts were seeded on plastic, immobilized HA or HC-HA/PTX3 or on plastic with or without soluble HA and HC-HA/PTX3 in DMEM+10% FBS, with or without AMD3100 or SB431542 in DMEM+ITS with or without transforming growth factor-ß1 (TGF-ß1). Transcript expression of keratocyte and signaling markers was determined by RT-qPCR. Immunostaining was performed to monitor cytolocalization of signaling markers and α-SMA. Western blotting was used to measure relative protein level. Results: Human corneal fibroblasts and myofibroblasts cultured in or on HC-HA/PTX3, but not HA, were refrained from cytoplasmic expression of αSMA and nuclear translocation of pSMAD2/3 when challenged with exogenous TGF-ß1. Such an antiscarring action by suppressing canonical TGF-ß1 signaling was surprisingly accompanied by phenotypic reversal to keratocan-expressing keratocytes through activation of BMP signaling. Further investigation disclosed that such phenotypic reversal was initiated by cell aggregation mediated by SDF1-CXCR4 signaling highlighted by nuclear translocation of CXCR4 and upregulation of CXCR4 transcript and protein followed by activation of canonical BMP signaling. Conclusions: These findings collectively provide mechanistic understanding explaining how amniotic membrane transplantation exerts an antiscarring action. In addition, HC-HA/PTX3 and derivatives may be developed into a new biologic to treat corneal blindness caused by stromal scar or opacity in the future.

One-year safety, healing and amputation rates of Wagner 3-4 diabetic foot ulcers treated with cryopreserved umbilical cord (TTAX01).

An open label, multicenter 16-week trial of cryopreserved human umbilical cord (TTAX01) was previously undertaken in 32 subjects presenting with a Wagner grade 3 or 4 diabetic foot ulcer, with 16 (50%) of these having confirmed closure following a median of one product application (previous study). All but two subjects (30/32; 94%) consented to participate in this follow-up study to 1-year postexposure. No restrictions were placed on treatments for open wounds. At 8-week intervals, subjects were evaluated for adverse events (AEs) and wound status (open or closed). Average time from initial exposure to end of follow-up was 378 days (range 343-433), with 29 of 30 (97%) subjects completing a full year. AEs were all typical for the population under study, and none were attributed to prior exposure to TTAX01. One previously healed wound re-opened, one previously unconfirmed closed wound remained healed, and nine new wound closures occurred, giving 25 of 29 (86.2%) healed in the ITT population. Three of the new closures followed the use of various tissue-based products. Three subjects whose wounds were healed required subsequent minor amputations due to osteomyelitis, one of which progressed to a major amputation (1/29; 3.4%). One additional subject underwent two minor amputations prior to healing. Overall, the study found TTAX01 to be safe in long-term follow-up and associated with both a low rate of major amputation and a higher than expected rates of healing.

Basic science review of birth tissue uses in ophthalmology.

The birth tissue is predominantly comprised of amniotic membrane (AM) and umbilical cord (UC), which share the same cell origin as the fetus. These versatile biological tissues have been used to treat a wide range of conjunctival and corneal conditions since 1940. The therapeutic benefits of the birth tissue stem from its anti-inflammatory and anti-scarring properties that orchestrate regenerative healing. Although the birth tissue also contains many cytokines, growth factors, and proteins, the heavy chain 1-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) matrix has been identified to be a major active tissue component responsible for AM/UC's multifactorial therapeutic actions. HC-HA/PTX3 complex is abundantly present in fresh and cryopreserved AM/UC, but not in dehydrated tissue. In this review, we discuss the tissue anatomy, the molecular mechanism of action based on HC-HA/ PTX3 to explain their therapeutic potentials, and the various forms available in ophthalmology.

Amniotic membrane transplantation for managing dry eye and neurotrophic keratitis.

Neurotrophic keratitis (NK), a degenerative disease caused by damage to the trigeminal nerve, abolishes both tearing and blinking reflexes, thus causing the most severe forms of dry eye disease (DED). Conversely, the increasing severity of DED also leads to progressive loss of corneal nerve density, potentially resulting in NK. Both diseases manifest the same spectrum of corneal pathologies including inflammation and corneal epithelial keratitis, which can progress into vision-threatening epithelial defect and stromal ulceration. This review summarizes the current literature regarding outcomes following sutured and sutureless cryopreserved amniotic membrane (AM) in treating DED as well as epithelial defects and corneal ulcers due to underlying NK. These studies collectively support the safety and effectiveness of cryopreserved AM in restoring corneal epithelial health, improving visual acuity in eyes with NK and DED, and alleviating symptomatic DED. Future randomized controlled trials are warranted to validate the above findings and determine whether such clinical efficacy lies in promoting corneal nerve regeneration.

Cryopreserved human umbilical cord versus acellular dermal matrix patches for in utero fetal spina bifida repair in a pregnant rat model.

OBJECTIVE: Despite significant improvement in spinal cord function after in utero spina bifida (SB) repair compared with traditional postnatal repair, over half of the children who undergo this procedure do not benefit completely. This lack of benefit has been attributed to closure methods of the defect, with subsequent spinal cord tethering at the repair site. Hence, a regenerative patch or material with antiinflammatory and anti-scarring properties may alleviate comorbidities with improved outcomes. The authors' primary objective was therefore to compare cryopreserved human umbilical cord (HUC) versus acellular dermal matrix (ADM) patches for regenerative repair of in utero SB lesions in an animal model. METHODS: In vivo studies were conducted in retinoic acid-induced SB defects in fetuses of Sprague-Dawley rats. HUC or ADM patches were sutured over the SB defects at a gestational age of 20 days. Repaired SB defect tissues were harvested after 48-52 hours. Tissue sections were immunofluorescently stained for the presence of neutrophils, macrophages, keratinocytes, meningeal cells, and astrocytes and for any associated apoptosis. In vitro meningeal or keratinocyte cell coculture experiments with the ADM and HUC patches were performed. All experiments were scored quantitatively in a blinded manner. RESULTS: Neutrophil counts and apoptotic cells were lower in the HUC-based repair group (n = 8) than in the ADM patch repair group (n = 7). In the HUC patch repair group, keratinocytes were present on the outer surface of the patch, meningeal cells were present on the inner surface of the patch adjacent to the neural placode, and astrocytes were noted to be absent. In the ADM patch repair group, all 3 cell types were present on both surfaces of the patch. In vitro studies showed that human meningeal cells grew preferentially on the mesenchymal side of the HUC patch, whereas keratinocytes showed tropism for the epithelial side, suggesting an inherent HUC-based cell polarity. In contrast, the ADM patch studies showed no polarity and decreased cellular infiltration. CONCLUSIONS: The HUC patch demonstrated reduced acute inflammation and apoptosis together with superior organization in regenerative cellular growth when compared with the ADM patch, and is therefore likely the better patch material for in utero SB defect repair. These properties may make the HUC biomaterial useful as a "meningeal patch" during spinal cord surgeries, thereby potentially reducing tethering and improving on spinal cord function.

An open-label trial of cryopreserved human umbilical cord in the treatment of complex diabetic foot ulcers complicated by osteomyelitis.

Clinical trials of potential new therapies for diabetic foot ulcers rarely enroll patients whose wounds extend to muscle, fascia, or bone with clinical and radiographic evidence of underlying osteomyelitis. An open-label, multicenter trial of cryopreserved human umbilical cord (TTAX01) was undertaken in 32 subjects presenting with such complex wounds with a mean duration of 6.1 ± 9.0 (range: 0.2-47.1) months and wound area at screening of 3.8 ± 2.9 (range: 1.0-9.6) cm2 . Aggressive surgical debridement at baseline resulted in 17 minor amputations and an increase in mean wound area to 7.4 ± 5.8 (range: 1.1-28.6) cm2 . All subjects were placed on systemic antibiotics for at least 6 weeks in conjunction with baseline application of TTAX01. Repeat applications were made at no less than 4-week intervals over the 16-week trial. Initial closure occurred in 18 of 32 (56%) wounds, with 16 (50%) of these having confirmed closure in 16 weeks with a median of one-product application. Cases with biopsy confirmed osteomyelitis (n = 20) showed initial closure in 12 (60%) wounds and confirmed closure in 10 (50%) wounds. Four of the five ulcers presenting as recurrences experienced confirmed closure. Mean overall time to healing was 12.8 ± 4.3 weeks. Mean wound area reduction from baseline was 91% for all wounds. Of the 16 wounds without confirmed closure during the 16-week treatment period, five (31.3%) achieved 99-100% wound area reduction by their final visit. The product was well tolerated. Two minor amputations occurred during the study period due to recurrent or persistent osteomyelitis; however, there were no major amputations.

Pax 6 Controls Neural Crest Potential of Limbal Niche Cells to Support Self-Renewal of Limbal Epithelial Stem Cells.

On ocular surface, corneal epithelial stem cells (SC) reside in limbus between cornea and conjunctiva. Pax6, an evolutionally conserved transcription factor essential for eye development, is expressed in post-natal corneal and limbal epithelia progenitors (LEPC) but not in underlying stroma. Because Pax6 is transiently expressed in developing corneal stroma and a subset of limbal and corneal stromal progenitors, we examined the role of Pax6 in limbal niche cells (LNC) in maintaining the phenotype of neural crest (NC) progenitors to support LEPC. Our results showed that nuclear Pax6 staining was found in freshly isolated LNC but not corneal stromal cells. Serial passaged LNC resulted in gradual loss of nuclear Pax6 (46 kDa) staining and neural crest progenitor status defined by the expression of embryonic SCs and NC markers, neurosphere formation, and differentiation into neurons, oligodendrocytes and astrocytes. Gain of function of 46 kDa Pax6 in late-passaged LNC resulted in nuclear Pax6 staining and promotion of the aforementioned NC progenitor status. In an in vitro reunion assay, early passaged LNC and late passaged LNC with overexpression of Pax6 inhibited the expression of corneal epithelial differentiation marker and promoted holoclone by LEPC. Therefore, expression of nuclear 46 kDa Pax6 in LNC plays an important developmental role in maintaining NC progenitor status to support self-renewal of corneal epithelial SCs in the limbal niche.

Safety and efficacy of 4-terpineol against microorganisms associated with blepharitis and common ocular diseases.

OBJECTIVE: Microbial infection has been reported to cause blepharitis, conjunctivitis and keratitis. We evaluated the safety and efficacy of a foam formulation of 2% 4-terpineol (T4O) against common ocular microorganisms. MATERIAL AND METHODS: The antimicrobial effect of a 2% T4O formulation was evaluated by the United States Pharmacopeia 51 (USP <51>) antimicrobial effectiveness test for 14 and 28 days, as well as by a Time Kill Study (ASTM E2315) with a 60 s exposure time. Its potential of causing skin and ocular irritation was evaluated by the Repeated Insult Patch Test and the Hen's Egg Chorioallantoic Membrane Test, respectively. RESULTS AND DISCUSSION: It was seen that 2% T4O formulation did not cause ocular irritation, skin irritation, sensitisation or allergic contact dermatitis in human subjects. Most importantly, it killed microorganisms listed in USP <51> at both 14 and 28 days and exerted a rapid killing effect within 60 s against 13 bacteria, 1 fungus and Acanthamoeba castellanii. CONCLUSION: The above finding suggests that 2% T4O formulation is safe and effective in killing microorganisms related to common ocular and skin infective diseases. TRANSLATIONAL RELEVANCE: Although the clinical efficacy in treating ocular disease was not directly studied; this foam formulation containing 2% T4O, based on the in vitro results of this work, demonstrated that it can potentially be used as a preservative-free cleansing agent for ocular hygiene maintenance due to its ability to exert a broad-spectrum antimicrobial effect without causing ocular or skin irritation.

Correction to: Columbid circoviruses detected in free ranging pigeons from Southern Brazil: insights on PiCV evolution.

Unfortunately, the word "evolution" was found missing in title of the original article which is corrected here by this erratum. The original article has been corrected.