Ophthalmic features and management of 86 patients with cryptophthalmos-A refined classification to assist in surgical planning.

BACKGROUND/AIMS: This study aimed to describe a cohort of patients with cryptophthalmos (CO), characterize associated oculofacial abnormalities, and expand the classification to summarize surgical strategies for managing CO. METHODS: A retrospective, interventional case series was conducted on 86 patients (124 eyes) with CO. The study proposed further classifying complete and incomplete CO into cyst, microphthalmia, anophthalmia, and normal eyeball based on globe structures and then modifying surgery accordingly. The demography, ophthalmic features, systemic anomalies, operation methods, and treatment outcomes were reviewed. RESULTS: CO was complete in seven eyes (5.6%) and incomplete in eight eyes (6.5%). A total of 109 eyes (87.9%) of abortive CO were encountered. Among 15 eyes (13 patients) of complete and incomplete types, 9 (60.0%) eyeballs were identified as cysts, 3 (20.0%) as microphthalmia, 1 (6.7%) as anophthalmia, and 2 (13.3%) as normal eyeballs. Cyst reduction was performed in eight eyes and one patient underwent enucleation with hydroxyapatite implantation. The socket was fit with an ocular prosthesis or a conformer after fornix and eyelid reconstruction. Microphthalmia was enucleated, and hydroxyapatites were implanted; patients were fit with ocular prosthesis or conformer after fornix and eyelid reconstruction. A complete CO with normal eyeball was reported with the eyesight of hand movement after ocular surface reconstruction. The upper eyelid contour and adequate fornix were maintained after coloboma repair and fornix reconstruction in all patients with abortive CO. CONCLUSION: This study demonstrates the clinical manifestations of different types of CO and expands the manifestation spectrum, proposing a refined classification of CO and modifying surgical strategies accordingly.

Insight into levofloxacin loaded biocompatible electrospun scaffolds for their potential as conjunctival substitutes.

The rehabilitation of visual acuity with severe conjunctival fibrosis depends on ocular reconstruction with suitable conjunctival substitutes. In this study, we have developed poly(lactic acid) (PLA) electrospun nanofibrous membranes (EFMs) surface coated by cellulose nanofibrils (CNF) and/or silk peptide (SP). The CNF coating improved the hydrophilicity and the SP coating proliferated conjunctival epithelial cells (CjECs). To prevent post-operative infections, the composite scaffolds were loaded with levofloxacin (LF), constantly exerting efficient bactericidal effects. In in vivo evaluations, the PLA EFMs presented excellent therapeutic effects by promoting structural and functional restoration of conjunctiva after transplant. Even with reduced topical administration of antibiotics, the coloboma treated with LF loaded scaffolds presented no infections. It could be deduced that the potent bacterial inhibition feature could save troubles for patients by minimizing the application of antibiotics post-surgery. Hence, the developed PLA EFMs loaded with LF could be promising conjunctival substitutes.

Human adipose-derived stem cells enriched with VEGF-modified mRNA promote angiogenesis and long-term graft survival in a fat graft transplantation model.

BACKGROUND: Fat grafting, as a standard treatment for numerous soft tissue defects, remains unpredictable and technique-dependent. Human adipose-derived stem cells (hADSCs) are promising candidates for cell-assisted therapy to improve graft survival. As free-living fat requires nutritional and respiratory sources to thrive, insufficient and unstable vascularization still impedes hADSC-assisted therapy. Recently, cytotherapy combined with modified mRNA (modRNA) encoding vascular endothelial growth factor (VEGF) has been applied for the treatment of ischemia-related diseases. Herein, we hypothesized that VEGF modRNA (modVEGF)-engineered hADSCs could robustly enhance fat survival in a fat graft transplantation model. METHODS: hADSCs were acquired from lipoaspiration and transfected with modRNAs. Transfection efficiency and expression kinetics of modRNAs in hADSCs were first evaluated in vitro. Next, we applied an in vivo Matrigel plug assay to assess the viability and angiogenic potential of modVEGF-engineered hADSCs at 1 week post-implantation. Finally, modVEGF-engineered hADSCs were co-transplanted with human fat in a murine model to analyze the survival rate, re-vascularization, proliferation, fibrosis, apoptosis, and necrosis of fat grafts over long-term follow-up. RESULTS: Transfections of modVEGF in hADSCs were highly tolerable as the modVEGF-engineered hADSCs facilitated burst-like protein production of VEGF in both our in vitro and in vivo models. modVEGF-engineered hADSCs induced increased levels of cellular proliferation and proangiogenesis when compared to untreated hADSCs in both ex vivo and in vivo assays. In a fat graft transplantation model, we provided evidence that modVEGF-engineered hADSCs promote the optimal potency to preserve adipocytes, especially in the long-term post-transplantation phase. Detailed histological analysis of fat grafts harvested at 15, 30, and 90 days following in vivo grafting suggested the release of VEGF protein from modVEGF-engineered hADSCs significantly improved neo-angiogenesis, vascular maturity, and cell proliferation. The modVEGF-engineered hADSCs also significantly mitigated the presence of fibrosis, apoptosis, and necrosis of grafts when compared to the control groups. Moreover, modVEGF-engineered hADSCs promoted graft survival and cell differentiation abilities, which also induced an increase in vessel formation and the number of surviving adipocytes after transplantation. CONCLUSION: This current study demonstrates the employment of modVEGF-engineered hADSCs as an advanced alternative to the clinical treatment involving soft-tissue reconstruction and rejuvenation.

Ophthalmological features and treatments in five cases of Waardenburg syndrome.

The present study aimed to investigate the ocular characteristics and treatment of Waardenburg syndrome (WS). A total of five patients with Waardenburg syndrome from our hospital, aged between 1 and 8 years, were included in the present study. The clinical data of these patients were analyzed, and the ocular manifestations and treatments were described. The general manifestations included hearing loss (1/5), broad high nasal root (2/5) and hypoplasia of alae nasi (2/5). Ophthalmological evaluations revealed ptosis (1/5), strabismus 1 (1/5), synophrys (2/5), telecanthus (5/5), iris hypopigmentation (5/5), high intraocular pressure (1/5) and choroidal hypopigmentation (1/5). For patients with characteristic external eye abnormalities, including ptosis, ocular plastic surgery was performed. For patients with only symptoms of iris heterochromia, no special treatment was required. The findings from the present study suggest that patients with WS may have several characteristic ocular manifestations. Abnormalities in the eyelid can be corrected by ocular plastic surgery, which is beneficial to children's physical and mental development.

Conjunctival reconstruction via enrichment of human conjunctival epithelial stem cells by p75 through the NGF-p75-SALL2 signaling axis.

Severe conjunctival diseases can cause significant conjunctival scarring, which seriously limits eye movement and affects patients' vision. Conjunctival reconstruction remains challenging due to the lack of efficient methods for stem cells enrichment. This study indicated that p75 positive conjunctival epithelial cells (CjECs) were mainly located in the basal layer of human conjunctival epithelium and showed an immature differentiation state in vivo. The p75 strongly positive (p75++) CjECs enriched by immuno-magnetic beads exhibited high expression of stem cell markers and low expression of differentiated keratins. During continuous cell passage cultivation, p75++ CjECs showed the strongest proliferation potential and were able to reconstruct the conjunctiva in vivo with the most complete structure and function. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs while proNGF played an opposite role. Altogether, p75++ CjECs present stem cell characteristics and exhibit the strongest proliferation potential so can be used as seed cells for conjunctival reconstruction, and NGF-p75-SALL2 signaling pathway was involved in regulating the differentiation of CjECs.

Evaluation of chronic ocular sequelae in patients with symblepharon caused by ocular burns.

AIM: To evaluate chronic ocular sequelae in patients with symblepharon caused by ocular burns and propose an objective grading system. METHODS: This was a retrospective, single-center clinical study. Patients with symblepharon caused by ocular burns at least six months later were assessed. Chronic ocular sequelae were classified into 3 categories (eyelid, conjunctiva, and cornea) and 9 chronic ocular sequelae [friction factors, exposure factors, conjunctival hyperemia, length of symblepharon, scope of adhesion, lacrimal area adhesion, loss of the palisades of Vogt (POV), corneal neovascularization, and corneal opacification]. Each ocular sequela was graded from 0 to 3, depending on the increasing severity. The 9 ocular sequelae were evaluated to obtain the total severity score for each eye. The total severity score was defined as Grade I (1-9), Grade II (10-18), and Grade III (19-27). Moreover, the correlation between the severity of chronic ocular sequelae and visual acuity, surgical strategy, and the prognosis was analyzed, respectively. RESULTS: Cases of 79 eyes with symblepharon caused by ocular burns were included in this study. Of these, 20 (25.32%) were defined as Grade I, 43 (54.43%) as Grade II, and 16 (20.25%) as Grade III. Eyes with a high total severity score had reduced visual acuity, required complicated surgery strategies, and poor prognosis (P<0.001). Multivariate regression analysis showed that the scope of adhesion, corneal opacification, and corneal neovascularization significantly affected visual acuity, surgical strategy, and prognosis (all P<0.001). CONCLUSION: The evaluation of chronic ocular sequelae enabled the development of an objective grading system for patients with symblepharon caused by ocular burns. This grading system can be applied to guide the treatment and predict the prognosis.

Exploitation of human mesenchymal stromal cell derived matrix towards the structural and functional restoration of the ocular surface.

Conjunctival restoration is indispensable to help maintain the ocular surface microenvironment by secreting lubricative mucins. However, conventional amniotic membrane transplantation therapy has many limitations in its application due to risks involved with disease transmission and alloreactivity. As decellularized tissues have been frequently used for tissue engineering and adipose mesenchymal stem cells (ADMSCs) have been acknowledged for their low immunogenicity, we fabricated a decellularized matrix of adipose-derived mesenchymal stromal cells (DMA) to study the therapeutic potential of DMA in healing conjunctival defects. In the present study, the fabricated DMA, with certain thickness, exhibited transplantation operability in vivo. When applied in conjunctival defect rabbit models, the sheet of DMA played a substantial role in providing structural support without causing cosmetic difference. Moreover, DMA displayed great superiority in promoting faster wound closure with better stratified epithelium containing more goblet cells than the amniotic membranes (AMs). Mechanistically, compared with the tissue culture plates (TCPs) and TCPs coated with collagen or fibronectin (two of the main components of DMA), DMA exhibited its unique property in maintaining the stem cells of CjECs in an undifferentiated state, which is highly essential for long-term conjunctival reconstruction. In addition, DMA effectively enhanced the proliferation of CjECs by activating stronger phosphorylation of the Akt signaling pathway, the results of which were further verified in the in vivo experiment via the histological examination of p-Akt levels in reconstructed conjunctival epithelium by DMA. Thus, the decellularized matrix of ADMSCs depicts a promising conjunctival substitute in ocular reconstruction.

Trehalose attenuates TGF-ß1-induced fibrosis of hSCFs by activating autophagy.

Conjunctival fibrosis is a process of extracellular matrix accumulation and the appearance of myofibroblasts in subconjunctival fibroblasts induced by injury or inflammation, which can significantly reduce the filtration efficiency of glaucoma filtration surgery. In this study, autophagy was confirmed to be involved in regulating the fibrosis of human subconjunctival fibroblasts (hSCFs) induced by TGF-ß1. Following the addition of rapamycin, we detected that autophagy activation could reduce the increased expression level of αSMA and the accumulation of extracellular matrix component proteins namely fibronectin and type I collagen induced by TGF-ß1 via the inhibition of SMAD2 phosphorylation. Following the addition of HCQ, the inhibition of autophagy aggravated TGF-ß1-induced fibrosis of hSCFs. We further verified that trehalose, a safe clinical drug, could alleviate TGF-ß1-induced fibrosis of hSCFs by activating autophagy and that these effects could be blocked by autophagy inhibition. In summary, autophagy was shown to be involved in the regulation of TGF-ß1-induced fibrosis of hSCFs, which provided a novel perspective for exploring the progression of this lesion. More importantly, the protective effects of trehalose on TGF-ß1-induced fibrosis of hSCFs were mediated by the activation of autophagy and could provide possible new approaches for the clinical treatment of conjunctival fibrosis.

Surface modified electrospun poly(lactic acid) fibrous scaffold with cellulose nanofibrils and Ag nanoparticles for ocular cell proliferation and antimicrobial application.

Corneal and conjunctival infections are common ocular diseases, sometimes, causing severe and refractory drug-resistant bacteria infections. Fungal keratitis is a leading cause for blindness and traditional medical treatment is unsatisfactory. Thus, there is an urge to develop a new therapy to deal with these cases. In this study, we developed surface modified poly(lactic acid) (PLA) electrospun nanofibrous membranes (EFMs) with silver nanoparticles (AgNPs) and cellulose nanofibrils (CNF) as scaffolds for cell proliferation and antimicrobial application. The AgNPs with a very low content (below 0.1%) were easily anchored on the surface of PLA EFMs by CNF, which endowed the scaffold with hydrophilicity and antibacterial ability. The in-vitro cell co-culture experiments showed that the scaffold had great biocompatibility to ocular epithelial cells, especially the scaffolds coated by CNF, which significantly proliferated cells. Furthermore, the antibacterial activity could reach >95% inhibiting Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) due to the implantation of AgNPs, and the antifungal activity was also outstanding with most of the Fusarium spp. inhibited. Hence, the developed PLA EFMs with CNF and AgNPs are promising ocular bandages to promote cell proliferation and kill infectious pathogens, exhibiting potential applications in ocular wound healing in the future.

Electrospun gelatin/PCL and collagen/PCL scaffolds for modulating responses of bone marrow endothelial progenitor cells.

The determination of potential transplantable substrates and substitution cells for corneal endothelium transplantation may compensate for the shortage of cornea donors. Appropriate biodegradable and biocompatible tissue-engineered substratum with seed cells for endothelial keratoplasty has been increasingly studied. In the present study, electrospun gelatin/polycaprolactone (PCL) and collagen/PCL scaffolds were successfully established. Bone marrow endothelial progenitor cells (BEPCs) were cultured on these scaffolds to determine whether the scaffolds may promote the proliferation of BEPCs as well as maintain stem cell characteristics. Two variations of hybrid scaffolds, collagen/PCL (70% collagen and 30% PCL) and gelatin/PCL (70% gelatin and 30% PCL), were established via electrospinning. Microscopic structure, hydrophilicity and wettability of the two scaffolds were subsequently investigated. BEPCs were separately cultured on the scaffolds and were also seeded on glass slides to establish the control group. Furthermore, cell morphology; adherence, as determined by investigation of F-actin expression levels; proliferation, as determined via Cell Counting Kit-8 assays, Ki-67 staining and bromodeoxyuridine (BrdU) staining; and stem cell markers, as determined by cluster of differentiation (CD)-34 and CD-133 protein expression levels; were investigated. In addition, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine gene expression. The two nanofiber scaffolds were established using electrospun techniques with expected hydrophilicity, wettability and biocompatibility. BEPCs were revealed to spread well on and strongly adhere to the collagen/PCL (70:30) and gelatin/PCL (70:30) scaffolds. Furthermore, Ki-67 and BrdU staining results revealed greater levels of positive dots on the two hybrid scaffolds compared with the control group. CD-34 and CD-133 protein staining demonstrated increased levels of fluorescence intensity on scaffolds compared with the control group. Furthermore, increased expression levels of differentiation markers, such as ATP binding cassette subfamily G member 2, leucine rich repeat containing G protein-coupled receptor 5 and CD166, were detected on both scaffolds. RT-qPCR results demonstrated that the expression of caspase-3, which is associated with apoptosis, was decreased on the two scaffolds compared with in the control group. The expression of inflammatory factors, including interleukin (IL)-1, exhibited a significant decrease on the gelatin/PCL scaffold compared with in the control group; whereas the difference between the expression level of IL-1 exhibited by the collagen/PCL group and the control group were not markedly different. Electrospun collagen/PCL and gelatin/PCL scaffolds exhibited the potential to enhance the adherence and proliferation of BEPCs. BEPCs cultured on the two scaffolds demonstrated increased stem cell characteristics and differentiation potential. Electrospun gelatin/PCL and collagen/PCL scaffolds may represent a promising substratum in tissue-engineered corneal endothelium.

Effects of corneal stromal cell- and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells.

AIM: To explore the effects of conditioned media on the proliferation of corneal endothelial cells (CECs) and to compare the efficiency of different conditioned media (CM). METHODS: Rat CECs, corneal stromal cells (CSCs), bone marrow-derived endothelial progenitor cells (BEPCs), and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and cultured in vitro. CM was collected from CSCs, BEPCs, and BMSCs. CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed. RESULTS: After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone-like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na(+)/K(+)-ATP, aquaporin 1 (AQP1), and zonula occludens 1 (ZO-1). Based on quantitative polymerase chain reaction (qPCR) analysis, Na(+)/K(+)-ATP expression in CSC-CM was notably upregulated by 1.3-fold (±0.036) (P<0.05, n=3). The expression levels of ZO-1, neuron specific enolase (NSE), Vimentin, paired homebox 6 (PAX6), and procollagen type VIII (COL8A1) were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation. CONCLUSION: CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation.

Reconstruction of conjunctival epithelium-like tissue using a temperature-responsive culture dish.

PURPOSE: To study the feasibility of engineering conjunctival epithelial cell sheets on a temperature-responsive culture dish for ocular surface reconstruction. METHODS: Rabbit conjunctival epithelial cells (rCjECs) were cultured in DMEM/F-12 (1:1) medium. The morphology and phenotype of the rCjECs were confirmed with phalloidin staining, periodic acid-Schiff (PAS) staining, and immunocytochemistry. The rCjECs cultured on a temperature-responsive culture dish for 10 days produced confluent conjunctival epithelial cell sheets. Then, the phenotype, structure, and function of the conjunctival epithelial cell sheets were examined. RESULTS: The conjunctival epithelial cells were compact, uniform, and cobblestone shape. All cultured conjunctival epithelial cells were harvested as intact cell sheets by reducing the culture temperature to 20 °C. Conjunctival epithelial cells were stratified in four to five cell layers similar to the conjunctival epithelium. CCK-8 analysis, 5-bromo-2'-deoxyuridine (BrdU) staining, and the live and dead viability assay confirmed that viable proliferation cells were retained in the cell sheets. Immunohistochemistry for CK4, CK19, and MUC5AC showed the cell sheets still maintained characteristics of the conjunctival epithelium. CONCLUSIONS: A temperature-responsive culture dish enables fabrication of viable conjunctival epithelial cell sheets with goblet cells and proliferative cells. Conjunctival epithelial cell sheets will be promising for reconstruction of the conjunctival epithelium.

Targeted transplantation of human umbilical cord blood endothelial progenitor cells with immunomagnetic nanoparticles to repair corneal endothelium defect.

Corneal endothelial dysfunction involves progressive corneal edema and loss of visual acuity, which result in the need for corneal transplantation. The global shortage of donor corneas limits the development of the surgery. Reconstruction of a bioengineered corneal endothelium might resolve this problem. Various scaffolds have been used, but poor biocompatibility and degradation limit their applications. In this study, a novel method of targeted cellular transplantation without permanent residence of cell carriers in the host was proposed. Human umbilical cord blood endothelial progenitor cells (UCB EPCs) were labeled with CD34 immunomagnetic nanoparticles. The efficiency of the magnet attraction was evaluated in vitro with a simple device simulating the anterior chamber. The UCB EPCs labeled with nanoparticles were transplanted into the anterior chamber of rabbits with magnet attraction. The results indicated that labeling the nanoparticles did not affect the proliferation of the UCB EPCs. The in vitro study indicated that the magnet could directionally attract UCB EPCs labeled with nanoparticles. The in vivo study indicated that the corneas in rabbits transplanted with UCB EPCs labeled with nanoparticles and magnet attraction became relatively transparent with little edema. These results showed that UCB EPCs labeled with CD34 immunomagnetic nanoparticles could be attracted directionally by a magnet and could repair corneal endothelial defects, providing a promising cell therapy for corneal endothelial dysfunction.

Adenosine triphosphate-induced rabbit corneal endothelial cell proliferation in vitro via the P2Y2-PI3K/Akt signaling axis.

PURPOSE: To investigate the effect of the ATP-P2Y2-PI3K/Akt signaling axis on promoting rabbit corneal endothelial cell (RCEC) proliferation in vitro. METHODS: Five concentrations of adenosine triphosphate (ATP; 1, 10, 25, 50 and 100 µM) were added to RCECs, and the cell proliferation was detected using Cell Counting Kit-8 (CCK8) and Ki67 immunohistochemical staining. Other P2Y2 receptor agonists and antagonists were added to the cells, and the proliferation effect was evaluated using CCK8 to determine the involvement of the P2Y2 receptor. Changes in the expression of phosphorylated Akt in RCECs treated with different concentrations of extracellular ATP and the duration of extracellular ATP on Akt phosphorylation were investigated using Western blotting. The pharmacological profiles with or without the PI3K/Akt pathway inhibitors were also determined using Western blotting. RESULTS: We found that 10 µM ATP strongly promoted RCEC proliferation in vitro. Additionally, 25 µM ATP had a proliferation effect, whereas other concentrations (1, 50 and 100 µM) had no effect compared with the control group. Selective P2Y2 receptor agonists (UTP, ATPγS and Ap4A) showed the same promotion effect, while P2Y2 antagonists and PI3K/Akt inhibitors inhibited the effect of ATP. Moreover, phosphorylated Akt could be induced by the addition of extracellular ATP at all five concentrations and lasted for 1 h. This phosphorylation was prevented by PI3K/Akt inhibitors and a P2Y2 antagonist. CONCLUSIONS: These findings showed that 10 µM ATP markedly promoted RCEC proliferation via the P2Y2-PI3K/Akt signaling axis.