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Eyelid plays a vital role in protecting the eye from injury or infection. Inflammation related eyelid diseases, such as blepharitis, are the most common ocular disorders that affect human's vision and quality of life. Due to the physiological barriers and anatomical structures of the eye, the bioavailability of topical administrated therapeutics is typically less than 5%. Herein, we developed a bio-responsive hydrogel drug delivery system using a generally recognized as safe compound, triglycerol monostearate (TG-18), for in-situ eyelid injection with sustained therapeutics release. In vitro, drug release and disassembly time of Rosiglitazone loaded hydrogel (Rosi-hydrogel) were estimated in the presence or absence of MMP-9, respectively. Moreover, the disassembly of TG-18 hydrogel was evaluated with 9-month-old and 12-month-old mice in vivo. Owing to the bio-responsive nature of Rosi-hydrogel, the on-demand Rosiglitazone release is achieved in response to local enzymes. These findings are proved by further evaluation in the age-related meibomian gland dysfunction mice model, and the bio-responsive hydrogel is used as an in-situ injection to treat eyelid diseases. Taken together, the in-situ eyelid injection with sustained drug release opens a window for the therapy of inflammation related eyelid diseases., Highlights • This study is the first application of injectable bio-responsive hydrogel for therapy of inflammation related eyelid diseases. • The enzyme response characteristic is extremely suitable for enhancing drug bioavailability in ocular drug delivery. • In-situ release of rosiglitazone can effectively treat age-related meibomian gland dysfunction in the mice model.

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., p75++ CjECs(conjunctival epithelial cells) were enriched by immuno‐magnetic beads and their stem cell properties were detected. After 10 days of culture on amniotic membrane, conjunctiva constructed by p75++/− CjECs was transplanted to a rabbit model of conjunctival defect to test the repair effect. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs.

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.

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

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-β1. 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-β1 via the inhibition of SMAD2 phosphorylation. Following the addition of HCQ, the inhibition of autophagy aggravated TGF-β1-induced fibrosis of hSCFs. We further verified that trehalose, a safe clinical drug, could alleviate TGF-β1-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-β1-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-β1-induced fibrosis of hSCFs were mediated by the activation of autophagy and could provide possible new approaches for the clinical treatment of conjunctival fibrosis.

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.

Silver nanoparticles have drawn considerable attention as excellent antimicrobial agents because of their characteristics, including broad antimicrobial spectrum, durable antimicrobial property, and large specific surface area. However, the toxicity of silver nanoparticles limits the in vivo application in the antimicrobial therapy field. Here, we developed a novel silver-based biomaterial to achieve favorable biocompatibility as well as enhanced antimicrobial activity. Silver microspheres (AgMPs) were synthesized using bovine serum albumin as a template and H2O2 as an activator. Electron microscopy results showed that AgMPs had a honeycombed inner structure with an approximate diameter of 800 nm. The minimum inhibitory concentration results exhibited that AgMPs had effective antimicrobial action against bacteria and fungi when the concentration was greater than 32 and 16 μg/mL, respectively. The cell proliferation results suggested that AgMPs have no influence on corneal epithelial cell growth when the concentration was under 25 μg/mL. The in vivo antifungal therapy experiments demonstrated that 25 μg/mL AgMPs could effectively combat Candida smooth wound infections. Overall, AgMPs exhibited substantial antimicrobial action on fungi in addition to biosafety on corneal epithelial cells at a concentration within 16-25 μg/mL. Our study shows that AgMPs can be used as an ocular surface drop candidate to treat fungal keratitis.

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.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.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.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.

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.

Additional file 1.

Electrospun hybrid nanofibers prepared using combinations of natural and synthetic polymers have been widely investigated in tissue engineering. In this study, silk fibroin (SF) and poly(L-lactic acid-co-e-caprolactone) (PLCL) hybrid scaffolds were successfully prepared by electrospinning. Scanning electron micrographs (SEM) showed that SF/PLCL scaffolds were composed of defect-free nanofibers with a smooth and homogeneous fiber morphology. Water contact angle measurements demonstrated that the scaffolds were hydrophilic. To assess the cell affinity of SF/PLCL scaffolds, rabbit conjunctival epithelial cells (rCjECs) were cultured on the electrospun scaffolds. Scanning electron micrographs and in vitro proliferation assays showed that the cells adhered and proliferated well on the scaffolds. The quantitative polymerase chain reaction (qPCR) results showed excellent expression of CjEC genes, with reduced expression of inflammatory mediators. Hematoxylin and eosin (H&E) staining showed that the engineered conjunctiva constructed with SF/PLCL scaffolds consisted of 2–4 layers of epithelium. Furthermore, SF/PLCL scaffolds transplanted subcutaneously exhibited excellent biocompatibility. Therefore, SF/PLCL scaffolds may find biomedical applications in conjunctival reconstruction in the near future.

Despite that poly (lactic acid) (PLA) has satisfactory biodegradation in vivo, its application in biomedicine is restricted due to its unsatisfactory cytocompatibility. Silk fiber (SF) has outstanding biocompatibility and silk fibroin protein obtained from silk by degumming has good hydrophilicity. Therefore, combining the PLA and silk can improve hydrophilicity of PLA to apply as biomedical materials. In this study, different concentrations of sodium hypochlorite (NaClO) were used to separate the silk to obtain multiscale silk fibers (MSFs), which were implanted into the PLA electrospun fibrous membranes (EFMs). The morphology and structure of silk fibers separated by different concentrations of NaClO were studied by Zetasizer Nano ZS, UV spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy. Moreover, the biocompatibility of the surface-modified PLA composite membranes by MSFs was investigated by cell cultivation and proliferation. The results showed that the surface-modified PLA EFMs through MSF bundles obtained from NaClO split silk exhibited a certain improvement on PLA hydrophilicity and enhancement on cellular compatibility, which could have a broad prospect in the practical application of biomedical materials.

Purpose To develop a new rabbit model of corneal endothelial injury using a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. Methods The corneal endothelia of 2 groups of New Zealand white rabbits were treated with an Nd:YAG laser in a uniformly scattered fashion. Rabbits in group A underwent laser burns on the whole corneal endothelium, including the limbus area, whereas rabbits in group B were subjected to laser burns in the central 9-mm diameter zone of the endothelium. Slit-lamp biomicroscopy, optical coherence tomography, applanation tonometry, confocal microscopy, scanning electron microscopy, and histological examinations were performed during 4 weeks of follow-up. Results In both groups, dotted or focal corneal endothelium defects were directly observed. The stroma was intact. Right after laser application, a series of clinical manifestations appeared, including subepithelial and stromal edema, increased central corneal thickness, and corneal opacity. Laser burn had more notable effects in group A than in group B. In both groups, we observed no damage to the intraocular structures, and intraocular pressure was normal after laser treatment. Conclusions Nd:YAG laser treatment in a 9-mm diameter zone of the endothelium can effectively induce bullous keratopathy in a rabbit, whereas treatment for the entire corneal endothelium maintains bullous keratopathy for a longer period. The procedure is simple and reproducible, and it retains normal intraocular structures. This study provided a promising model for future research into endothelial cell damage and for the development of new therapies.

PurposeThe objective of this study is to evaluate and grade the extent and severity of ocular involvement in Tessier number 10 cleft.Patients and methodsA retrospective, noncomparative, interventional case series was conducted between January 2006 and December 2015. Clinical data were reviewed from 59 patients (85 eyes) with Tessier number 10 clefts. Detailed medical history and ophthalmic examination of patients with a confirmed diagnosis of Tessier number 10 cleft were recorded on an itemized data collection form. Ocular manifestations were categorized as upper eyelid defect, symblepharon with cutaneous pterygium (skin growing onto the globe), corneal complications, and lower eyelid ectropion; components were evaluated and graded on a scale from 0 to 3, according to their severity.ResultsMore than half of the cases (43 eyes, 53.8%) had severe upper eyelid defect, and severe symblepharon with cutaneous pterygium were observed in 38 eyes (47.5%). Nearly half of the cases (40 eyes, 50.0%) have severe corneal complications, and lower eyelid ectropion was found in 34 eyes (42.5%). The severity of symblepharon, corneal complications, and lower eyelid ectropion were significantly correlated with the upper eyelid defect; the correlation coefficient (r) ranged from 0.844 to 0.629 (P

Purpose This study aimed to investigate the therapeutic effects and underlying mechanisms of locally delivered regulatory T cells (Tregs) on acute corneal wound healing after alkali burn. Methods After corneal alkali burn, the mice were injected subconjunctivally with regulatory T cells (Tregs) isolated from syngeneic mice. The wound healing process was monitored by clinical manifestation, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). As amphiregulin (Areg) was significantly upregulated, its reparative function in injured corneas was suggested. The hypothesis was further verified via loss- and gain-of-function experiments by administrating the antibody of Areg (anti-Areg) and recombinant Areg (rmAreg). Results Subconjunctivally injected Tregs rapidly migrated to injured corneas. The mice treated with Tregs showed prominently reduced corneal opacity, alleviated edema, and faster re-epithelialization compared with the control group. Mechanistically, Treg treatment led to suppressed infiltration of inflammatory cells, along with improved proliferation and inhibited apoptosis of corneal epithelial cells. Tregs expressed upregulated functional markers, including Areg. Expectantly, the levels of Areg in corneas were dramatically higher in the Treg injection group, in line with better corneal restoration. Additional experiments showed that the administration of anti-Areg blunted the reparative effect of Tregs, while exogenous Areg enhanced it. Treg-treated corneas also exhibited less neovascularization and fibrosis at a later reconstruction stage of corneal repair. Conclusions The findings showed that the subconjunctival injection of Tregs effectively promoted corneal wound healing by inhibiting excessive inflammation and enhancing epithelial regeneration, with an indispensable reparative role of Areg. Subsequent complications of corneal vascularization and fibrosis were therefore reduced.

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.

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.

Electrospun hybrid nanofibers prepared using combinations of natural and synthetic polymers have been widely investigated in tissue engineering. In this study, silk fibroin (SF) and poly(l-lactic acid

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

Biocompatible polymer scaffolds are promising as potential carriers for the delivery of retinal progenitor cells (RPCs) in cell replacement therapy for the repair of damaged or diseased retinas. The primary goal of the present study was to investigate the effects of blended electrospun nanofibrous membranes of silk fibroin (SF) and poly(L-lactic acid-co-ε-caprolactone) (PLCL), a novel scaffold, on the biological behaviour of RPCs in vitro. To assess the cell-scaffold interaction, RPCs were cultured on SF/PLCL scaffolds for indicated durations. Our data revealed that all the SF/PLCL scaffolds were thoroughly cytocompatible and the SF:PLCL (1:1) scaffolds yielded the best RPC growth. The in vitro proliferation assays showed that RPCs proliferated more quickly on the SF:PLCL (1:1) than on the other scaffolds and the control. Quantitative polymerase chain reaction (qPCR) and immunocytochemistry analyses demonstrated that RPCs grown on the SF:PLCL (1:1) scaffolds preferentially differentiated toward retinal neurons, including, most interestingly, photoreceptors. In summary, we demonstrated that the SF:PLCL (1:1) scaffolds can not only markedly promote RPC proliferation with cytocompatibility for RPC growth but also robustly enhance RPCs’ differentiation toward specific retinal neurons of interest in vitro, suggesting that SF:PLCL (1:1) scaffolds may have potential applications in retinal cell replacement therapy in the future.

Junzhao Chen,1,* Chenxi Yan,1,* Mengyu Zhu,1,* Qinke Yao,1 Chunyi Shao,1 Wenjuan Lu,1 Jing Wang,2 Xiumei Mo,2 Ping Gu,1 Yao Fu,1 Xianqun Fan1 1Department of Ophthalmology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 2Biomaterials and Tissue Engineering Laboratory, College of Chemistry and Chemical Engineering and Biotechnology, Donghua University, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: Cornea transplant technology has progressed markedly in recent decades, allowing surgeons to replace diseased corneal endothelium by a thin lamellar structure. A thin, transparent, biocompatible, tissue-engineered substratum with corneal endothelial cells for endothelial keratoplasty is currently of interest. Electrospinning a nanofibrous structure can simulate the extracellular matrix and have beneficial effects for cell culture. Silk fibroin (SF) has good biocompatibility but poor mechanical properties, while poly(L-lactic acid-co-Ɛ-caprolactone) (P(LLA-CL)) has good mechanical properties but poor biocompatibility. Blending SF with P(LLA-CL) can maintain the advantages of both these materials and overcome their disadvantages. Blended electrospun nanofibrous membranes may be suitable for regeneration of the corneal endothelium. The aim of this study was to produce a tissue-engineered construct suitable for endothelial keratoplasty.Methods: Five scaffolds containing different SF:P(LLA-CL) blended ratios (100:0, 75:25, 50:50, 25:75, 0:100) were manufactured. A human corneal endothelial (B4G12) cell line was cultured on the membranes. Light transmission, speed of cell adherence, cell viability (live-dead test), cell proliferation (Ki-67, BrdU staining), and cell monolayer formation were detected on membranes with the different blended ratios, and expression of some functional genes was also detected by real-time polymerase chain reaction.Results: Different blended ratios of scaffolds had different light transmittance properties. The 25:75 blended ratio membrane had the best transmittance among these scaffolds. All electrospun nanofibrous membranes showed improved speed of cell adherence when compared with the control group, especially when the P(LLA-CL) ratio increased. The 25:75 blended ratio membranes also had the highest cell proliferation. B4G12 cells could form a monolayer on all scaffolds, and most functional genes were also stably expressed on all scaffolds. Only two genes showed changes in expression.Conclusion: All blended ratios of SF:P(LLA-CL) scaffolds were evaluated and showed good biocompatibility for cell adherence and monolayer formation. Among them, the 25:75 blended ratio SF:P(LLA-CL) scaffold had the best transmittance and the highest cell proliferation. These attributes further the potential application of the SF:P(LLA-CL) scaffold for corneal endothelial transplantation. Keywords: silk fibroin, poly(L-lactic acid-co-Ɛ-caprolactone), B4G12, corneal endothelium, regeneration

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.

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.