David Hui-Kang Ma

Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane.

Purpose. To identify the potential antiangiogenic and antiinflammatory proteins expressed in human amniotic membrane tissue. Methods. Human amniotic epithelial and mesenchymal cells were isolated from human amniotic membranes by sequential trypsin and collagenase digestion. Total RNAs were harvested from freshly obtained human amniotic epithelial and mesenchymal cells. Antiangiogenic and antiinflammatory proteins were detected by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique and further confirmed by DNA sequencing of PCR-amplified transcripts. The distribution of tissue inhibitors of metalloproteinase (TIMPs) were studied further by immunohistochemistry performed on paraffin-embedded amniotic membrane tissue. Results. RT-PCR results showed that both human amniotic epithelial and mesenchymal cells express interleukin-1 receptor antagonist, all four TIMPs, collagen XVIII, and interleukin-10. Thrombospondin-1 was expressed in all of the epithelial cell specimens and in one out of five mesenchymal cell specimens. Furthermore, immunohistochemistry studies performed on freshly prepared amniotic membrane confirmed that all members of the TIMP family were present in epithelial and mesenchymal cells as well as in the compact layer of the amniotic stroma. In cryopreserved amniotic membranes, positive staining was seen in residual amniotic cells and stroma. Conclusions. Human amniotic membrane epithelial and mesenchymal cells express various antiangiogenic and antiinflammatory proteins. Some of those proteins also were found in amniotic membrane stroma. These findings may explain in part the antiangiogenic and antiinflammatory effects of amniotic membrane transplantation.

Amniotic membrane graft for primary pterygium: comparison with conjunctival autograft and topical mitomycin C treatment

AIM To study the efficacy and safety of amniotic membrane graft as an adjunctive therapy after removal of primary pterygium, and to compare the clinical outcome with conjunctival autograft and topical mitomycin C. METHODS 80 eyes of 71 patients with primary pterygia were treated with excision followed by amniotic membrane graft. The result was compared retrospectively with 56 eyes of 50 patients receiving conjunctival autograft, and 54 eyes of 46 patients receiving topical mitomycin C. Patients were followed for at least 6 months, and the averaged follow up periods for the three groups were 13.8, 22.8, and 18.4 months, respectively. RESULTS There were three recurrences (3.8%) in the amniotic membrane graft group, three recurrences (5.4%) in the conjunctival autograft group, and two recurrences (3.7%) in the topical mitomycin C group. There was no significant difference in recurrence rate among the three groups (p = 0.879). No major complications occurred in the amniotic membrane graft group or the conjunctival autograft group. One case of infectious scleritis due to scleral ischaemia occurred in the topical mitomycin C group. CONCLUSION This study showed that amniotic membrane graft was as effective as conjunctival autograft and mitomycin C in preventing pterygium recurrence, and can be considered as a preferred grafting procedure for primary pterygium.

Carbodiimide cross-linked amniotic membranes for cultivation of limbal epithelial cells.

In ophthalmic tissue engineering, amniotic membrane (AM) is one of the most prevalent natural matrices used for limbal epithelial cell (LEC) cultivation and transplantation. However, the application of AM as a scaffold is limited by its low biomechanical strength and rapid biodegradation. The present study reports the development of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linked AM as an LEC carrier. The collagenous tissue materials were modified with varying cross-linker concentrations (0-0.25 mmol EDC/mg AM) and were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), ninhydrin assays, electron microscopy, light transmission measurements, mechanical and in vitro degradation tests, as well as diffusion permeability and cell culture studies. Our results showed that chemical cross-linking approaches saturation at concentrations of 0.05 mmol EDC/mg AM. The formation of cross-links (i.e., amide bonds) in the samples treated with 0.05 mmol EDC/mg AM may cause significant aggregation of tropocollagen molecules and collagen microfibrils without affecting cell morphology of biological tissues. With the optimum concentration of 0.05 mmol EDC/mg AM, chemical cross-linker could significantly enhance the mechanical and thermal stability, optical transparency, and resistance to collagenase digestion. Continuous permeation of albumin through the cross-linked AM would be helpful to cell growth over the matrix surface. In addition, the EDC cross-linked samples were able to support LEC proliferation and preserve epithelial progenitor cells in vitro and in vivo. It is concluded that the AM cross-linked with 0.05 mmol EDC/mg AM may be a potential biomaterial for regenerative medicine.

Carbodiimide cross-linked hyaluronic acid hydrogels as cell sheet delivery vehicles: characterization and interaction with corneal endothelial cells.

It was reported that cell-adhesive gelatin discs have been successfully used as delivery vehicles for intraocular grafting of bioengineered corneal endothelial cell sheets. Development of alternative biomaterials to bovine-based gelatin vehicles can potentially eliminate the risk of bovine spongiform encephalopathy. In the present work, to investigate whether it was appropriate for use as cell sheet delivery vehicles, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) cross-linked hyaluronic acid (HA) hydrogels were studied by determinations of morphological characteristic, mechanical and thermal property, water content, in vitro degradability and cytocompatibility. Glutaraldehyde (GTA) cross-linked HA samples were used for comparison. It was found that HA discs after cross-linking significantly increased its tensile stress but reduced its tensile strain, water uptake and enzymatic degradability. The results of differential scanning calorimetry demonstrated that cross-linking could lead to the alteration of polymer structure. In addition, the EDC-cross-linked HA discs had a smoother surface structure, a faster degradation rate and a relatively lower cytotoxicity as compared to the GTA cross-linked counterparts. It is concluded that EDC can be successfully applied for HA cross-linking to fabricate structurally stable, mechanically reinforced, readily deformable, transparent and cytocompatible HA hydrogel discs with the potential to be applied as delivery vehicles for corneal endothelial cell therapy.

Ocular Biocompatibility of Carbodiimide Cross-Linked Hyaluronic Acid Hydrogels for Cell Sheet Delivery Carriers

Due to its innocuous nature, hyaluronic acid (HA) is one of the most commonly used biopolymers for ophthalmic applications. We recently developed a cell sheet delivery system using carbodiimide cross-linked HA carriers. Chemical cross-linking provides an improvement in stability of polymer gels, but probably causes toxic side-effects. The aim of this study was to investigate the ocular biocompatibility of HA hydrogels cross-linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). HA discs without cross-linking and glutaraldehyde (GTA) cross-linked HA samples were used for comparison. The disc implants were inserted in the anterior chamber of rabbit eyes for 24 weeks and characterized by slit-lamp biomicroscopy, histology and scanning electron microscopy. The ophthalmic parameters obtained from biomicroscopic examinations were also scored to provide a quantitative grading system. Results of this study showed that the HA discs cross-linked with EDC had better ocular biocompatibility than those with GTA. The continued residence of GTA cross-linked HA implants in the intraocular cavity elicited severe tissue responses and significant foreign body reactions, whereas no adverse inflammatory reaction was observed after contact with non-cross-linked HA or EDC cross-linked HA samples. It is concluded that the cross-linking agent type gives influence on ocular biocompatibility of cell carriers and the EDC-HA hydrogel is an ideal candidate for use as an implantable material in cell sheet delivery applications.

Persistence of Transplanted Oral Mucosal Epithelial Cells in Human Cornea

PURPOSE To determine the expression of differentiation and progenitor cell markers in corneal tissues that previously underwent autologous cultivated oral mucosal epithelial transplantation (COMET). METHODS Four eyes from three alkaline-injured patients and one thermally injured patient underwent COMET to promote re-epithelialization or corneal reconstruction. Between 10 and 22 months (mean, 14.2 +/- 5.5 months [SD]) after COMET, the corneal tissues were obtained after penetrating keratoplasty (n = 1) or autologous limbal transplantation (n = 3). Immunoconfocal microscopy for keratin (K)3, -12, -4, -13, and -8; connexin (Cx)43; MUC5AC; laminin-5; pan-p63; ABCG2; and p75 was performed in those specimens as well as in the oral mucosa and cultivated oral mucosal epithelial cells (OMECs). RESULTS All four specimens were unanimously positive for K3, -4, and -13 but negative for K8 and MUC5AC, suggesting that the keratinocytes were oral mucosa-derived. However, peripheral K12 staining was positive only in patient 2, suggesting a mixed oral and corneal epithelium in that case. Cx43 staining in the basal epithelium was negative in patients 1, 2, and 3, but was positive in patient 4. Small, compact keratinocytes in the basal epithelium preferentially expressed pan-p63, ABCG2, and p75. Although the staining of pan-p63 and ABCG2 tended to be more than one layer, signal for p75 was consistently localized only to the basal layer. CONCLUSIONS The study demonstrated the persistence of transplanted OMECs in human corneas. In addition, small, compact cells in the basal epithelium preferentially expressed the keratinocyte stem/progenitor cell markers, which may be indicative of the engraftment of the progenitor cells after transplantation.

Characterization of Cross-Linked Porous Gelatin Carriers and Their Interaction with Corneal Endothelium: Biopolymer Concentration Effect

Cell sheet-mediated tissue regeneration is a promising approach for corneal reconstruction. However, the fragility of bioengineered corneal endothelial cell (CEC) monolayers allows us to take advantage of cross-linked porous gelatin hydrogels as cell sheet carriers for intraocular delivery. The aim of this study was to further investigate the effects of biopolymer concentrations (5–15 wt%) on the characteristic and safety of hydrogel discs fabricated by a simple stirring process combined with freeze-drying method. Results of scanning electron microscopy, porosity measurements, and ninhydrin assays showed that, with increasing solid content, the pore size, porosity, and cross-linking index of carbodiimide treated samples significantly decreased from 508±30 to 292±42 µm, 59.8±1.1 to 33.2±1.9%, and 56.2±1.6 to 34.3±1.8%, respectively. The variation in biopolymer concentrations and degrees of cross-linking greatly affects the Young’s modulus and swelling ratio of the gelatin carriers. Differential scanning calorimetry measurements and glucose permeation studies indicated that for the samples with a highest solid content, the highest pore wall thickness and the lowest fraction of mobile water may inhibit solute transport. When the biopolymer concentration is in the range of 5–10 wt%, the hydrogels have high freezable water content (0.89–0.93) and concentration of permeated glucose (591.3–615.5 µg/ml). These features are beneficial to the in vitro cultivation of CECs without limiting proliferation and changing expression of ion channel and pump genes such as ATP1A1, VDAC2, and AQP1. In vivo studies by analyzing the rabbit CEC morphology and count also demonstrate that the implanted gelatin discs with the highest solid content may cause unfavorable tissue-material interactions. It is concluded that the characteristics of cross-linked porous gelatin hydrogel carriers and their triggered biological responses are in relation to biopolymer concentration effects.

Comparison of amniotic membrane graft alone or combined with intraoperative mitomycin C to prevent recurrence after excision of recurrent pterygia

Purpose: Without effective adjunctive therapy, the recurrence rate after excision of recurrent pterygia is high. In an effort to determine a way to better reduce the recurrence of pterygia, we compared the efficacy and safety of amniotic membrane graft (AMG) alone and AMG combined with intraoperative mitomycin C after excision of recurrent pterygia. Methods: Patients with recurrent pterygia were randomly assigned to receive excision of pterygia followed by AMG alone or AMG combined with intraoperative 0.025% mitomycin C for 3 minutes (AMG-mitomycin C). Patients who could be followed up for more than 12 months were entered for data analysis. Conjunctival (potential) and corneal (true) recurrence of pterygia and other complications were recorded. Results: The group receiving AMG alone was made up of 48 eyes in 48 patients, and the group receiving AMG-mitomycin C consisted of 47 eyes in 46 patients. No significant difference was found in age distribution, sex distribution, or duration of follow-up between the 2 groups. Regarding the recurrence rates of each group, 6 conjunctival (12.5%) and 6 corneal (12.5%) recurrences developed in the AMG group, and 4 conjunctival (8.5%) and 6 corneal (12.8%) recurrences developed in the AMG-mitomycin C group. No significant difference was found in the conjunctival and corneal recurrence rate between the 2 groups (P = 0.623 and 0.966, respectively; log rank test), and no major complications developed in either group. Conclusions: AMG alone can be considered an effective alternative adjunctive treatment of recurrent pterygia. The addition of intraoperative mitomycin C did not further reduce the recurrence rate.

Regulation of corneal angiogenesis in limbal stem cell deficiency

Corneal angiogenesis is associated with a variety of corneal diseases, and is sometimes vision threatening. In recent years, with the discovery of major pro- and anti-angiogenic factors in the cornea, details of the angiogenic process are gradually unveiled. Of note, corneal inflammation and neovascularization associated with severe limbal stem cell (LSC) deficiency is a clinically challenging issue in that the condition persists long after the initial insult, and will not improve without transplantation of LSCs. However, to date the molecular mechanism by which LSC transplantation restores corneal avascularity is not fully understood. In addition to discussing major pro-angiogenic factors involved in corneal neovascularization, this review article also focuses on possible molecular mechanisms underlying persistent inflammation and neovascularization following severe LSC deficiency, and anti-angiogenic factors expressed by human limbo-corneal epithelial cells (HLCECs). Most of the recently discovered corneal anti-angiogenic factors belong to extracellular matrix proteins that acquire angio-inhibitory activity only after proper proteolytic processing. Our recent findings showed that the secretion of endostatin (derived from basement membrane collagen XVIII) and restin (from collagen XV) by HLCECs were enhanced when HLCECs were cultivated on amniotic membrane (AM). This adds to the advantage of transplanting ex vivo expanded HLCECs cultivated on AM in that the anti-angiogenic activity of the epithelial cells is augmented in a physiological way. Furthermore, proteomic profiling of HLCECs and human conjunctival epithelial cells (HCECs) identified a 14-3-3 protein (stratifin) preferentially expressed by HLCECs. In addition to functioning as a cell cycle controller, keratinocyte-derived stratifin induces MMPs which are involved in the generation of restin (by MMP-1) and endostatin (by MMP-3). These findings highlight the significance of delicate epithelial-matrix interactions in the maintenance of corneal avascularity.

Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis.

The purpose of this study is to demonstrate the application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for the ex-vivo visualization of human corneal morphological alterations due to infectious processes. The structural alterations of both cellular and collagenous components can be respectively demonstrated using fluorescence and SHG imaging. In addition, pathogens with fluorescence may be identified within turbid specimens. Our results show that multiphoton microscopy is effective for identifying structural alterations due to corneal infections without the need of histological processing. With additional developments, multiphoton microscopy has the potential to be developed into an imaging technique effective in the clinical diagnosis and monitoring of corneal infections.