Gary Hin-Fai Yam

Ex Vivo Propagation of Human Corneal Stromal "Activated Keratocytes" for Tissue Engineering.

Keratoconus is a corneal disorder characterized by a thinning of stromal tissue, and the affected patients have induced astigmatism and visual impairment. It is associated with a loss of corneal stromal keratocytes (CSKs). Hence, reconstructing stromal tissue with autologous CSK replacement can be a viable alternative to corneal transplantation, which is restricted by the global donor material shortage and graft rejection. Human CSKs are normally quiescent and express unique markers, like aldehyde dehydrogenases and keratocan. In serum culture, they proliferate, but lose their characteristic phenotype and become stromal fibroblasts. Here we report a novel culture cocktail to ex vivo propagate and maintain CSKs. Primary human CSKs were obtained from adult donors and cultured with soluble human amnion stromal extract (ASE), rho-associated coiled-coil-forming protein serine/threonine kinase inhibitor Y-27632, and insulin-like growth factor-1 (collectively named as ERI). Protein profiling using mass spectrometry followed by MetaCore” pathway analysis predicted that ASE proteins might participate in transforming growth factor-β (TGF-β) signaling and fibroblast development, cell adhesion, extracellular matrix remodeling, and immune response. In culture with 0.5% fetal bovine serum and ERI, the population of “activated keratocytes” was expanded. They had much lowered expression of both keratocyte and fibroblast markers, suppressed TGF-β-mediated Smad2/3 activation, and lacked fibroblast-mediated collagen contractibility. These “activated keratoctyes” could be propagated for six to eight passages ex vivo, and they regained CSK-specific dendritic morphology and gene marker expression, including aldehyde dehydrogenases, lumican, and keratocan biosynthesis, expression, and secretion when returned to serum-depleted ERI condition. This novel cocktail maintained human CSKs in both adherent and suspension cultures with proper keratocyte features and without the transformation to stromal fibroblasts. Thus, human CSKs can be ex vivo propagated as transient “activated keratocytes.” This could provide sufficient number of genuine CSKs for corneal tissue engineering.

Altered Anterior Segment Biometric Parameters in Mice Deficient in SPARC

Purpose Secreted protein acidic and rich in cysteine (SPARC) and Hevin are structurally related matricellular proteins involved in extracellular matrix assembly. In this study, we compared the anterior chamber biometric parameters and iris collagen properties in SPARC-, Hevin- and SPARC-/Hevin-null with wild-type (WT) mice. Methods The right eyes of 53 WT, 35 SPARC-, 56 Hevin-, and 63 SPARC-/Hevin-null mice were imaged using the RTVue-100 Fourier-domain optical coherence tomography system. The parameters measured were anterior chamber depth (ACD), trabecular-iris space area (TISA), angle opening distance (AOD), and pupil diameter. Biometric data were analyzed using analysis of covariance and adjusted for age, sex, and pupil diameter. Expression of Col1a1, Col8a1, and Col8a2 transcripts in the irises was measured by quantitative polymerase chain reaction. Collagen fibril thickness was evaluated by transmission electron microscopy. Results Mice that were SPARC- and SPARC-/Hevin-null had 1.28- and 1.25-fold deeper ACD, 1.45- and 1.53-fold larger TISA, as well as 1.42- and 1.51-fold wider AOD than WT, respectively. These measurements were not significantly different between SPARC- and SPARC-/Hevin-null mice. The SPARC-null iris expressed lower Col1a1, but higher Col8a1 and Col8a2 transcripts compared with WT. Collagen fibrils in the SPARC- and SPARC-/Hevin-null irises were 1.5- and 1.7-fold thinner than WT, respectively. The Hevin-null iris did not differ from WT in these collagen properties. Conclusions SPARC-null mice have deeper anterior chamber as well as wider drainage angles compared with WT. Therefore, SPARC plays a key role in influencing the spatial organization of the anterior segment, potentially via modulation of collagen properties, while Hevin is not likely to be involved.

Postnatal periodontal ligament as a novel adult stem cell source for regenerative corneal cell therapy

Corneal opacities are a leading cause of global blindness. They are conventionally treated by the transplantation of donor corneal tissue, which is, restricted by a worldwide donor material shortage and allograft rejection. Autologous adult stem cells with a potential to differentiate into corneal stromal keratocytes (CSKs) could offer a suitable choice of cells for regenerative cell therapy. Postnatal periodontal ligament (PDL) contains a population of adult stem cells, which has a similar embryological origin as CSK, that is cranial neural crest. We harvested PDL cells from young adult teeth extracted because of non‐functional or orthodontic reason and differentiated them towards CSK phenotype using a two‐step protocol with spheroid formation followed by growth factor and cytokine induction in a stromal environment (human amnion stroma and porcine corneal stroma). Our results showed that the PDL‐differentiated CSK‐like cells expressed CSK markers (CD34, ALDH3A1, keratocan, lumican, CHST6, B3GNT7 and Col8A2) and had minimal expression of genes related to fibrosis and other lineages (vasculogenesis, adipogenesis, myogenesis, epitheliogenesis, neurogenesis and hematogenesis). Introduction of PDL spheroids into the stroma of porcine corneas resulted in extensive migration of cells inside the host stroma after 14‐day organ culture. Their quiescent nature and uniform cell distribution resembled to that of mature CSKs inside the native stroma. Our results demonstrated the potential translation of PDL cells for regenerative corneal cell therapy for corneal opacities.

Safety and Feasibility of Intrastromal Injection of Cultivated Human Corneal Stromal Keratocytes as Cell-Based Therapy for Corneal Opacities

Purpose To evaluate the safety and feasibility of intrastromal injection of human corneal stromal keratocytes (CSKs) and its therapeutic effect on a rodent early corneal opacity model. Methods Twelve research-grade donor corneas were used in primary culture to generate quiescent CSKs and activated stromal fibroblasts (SFs). Single and repeated intrastromal injections of 2 to 4 × 104 cells to rat normal corneas (n = 52) or corneas with early opacities induced by irregular phototherapeutic keratectomy (n = 16) were performed, followed by weekly examination of corneal response under slit-lamp biomicroscopy and in vivo confocal microscopy with evaluation of haze level and stromal reflectivity, and corneal thickness using anterior segment optical coherence tomography (AS-OCT). Time-lapse tracing of Molday ION-labelled cells was conducted using Spectralis OCT and label intensity was measured. Corneas were collected at time intervals for marker expression by immunofluorescence, cell viability, and apoptosis assays. Results Injected CSKs showed proper marker expression with negligible SF-related features and inflammation, hence maintaining corneal clarity and stability. The time-dependent loss of injected cells was recovered by repeated injection, achieving an extended expression of human proteoglycans inside rat stroma. In the early corneal opacity model, intrastromal CSK injection reduced stromal reflectivity and thickness, resulting in recovery of corneal clarity, whereas noninjected corneas were thicker and had haze progression. Conclusions We demonstrated the safety, feasibility, and therapeutic efficacy of intrastromal CSK injection. The cultivated CSKs can be a reliable cell source for potential cell-based therapy for corneal opacities.

Reshaping and Customization of SMILE-Derived Biological Lenticules for Intrastromal Implantation

Purpose To evaluate the feasibility of excimer laser reshaping of biological lenticules available after small incision lenticule extraction (SMILE). Methods Fresh and cryopreserved SMILE-derived human lenticules underwent excimer laser ablation for stromal reshaping. The treatment effects in the lasered group were compared with the nonlasered group with respect to changes in surface functional groups (by Fourier transform infrared spectroscopy [FTIR]) and surface morphology (by scanning electron microscopy [SEM] and atomic force microscopy [AFM]). Ten SMILE-derived porcine lenticules, five nonlasered (107-μm thick, -6 diopter [D] spherical power) and five excimer lasered (50% thickness reduction), were implanted into a 120-μm stromal pocket of 10 porcine eyes. Corneal thickness and topography were assessed before and after implantation. Results FTIR illustrated prominent changes in the lipid profile. The collagen structure was also affected by the laser treatment but to a lesser extent. SEM exhibited a more regular surface for the lasered lenticules, confirmed by the lower mean Rz value (290.1 ± 96.1 nm vs. 380.9 ± 92.6 nm, P = 0.045) on AFM. The lasered porcine lenticules were thinner than the nonlasered controls during overhydration (132 ± 26 μm vs. 233 ± 23 μm, P < 0.001) and after 5 hours in a moist chamber (46 ± 3 μm vs. 57 ± 3 μm, P < 0.001). After implantation, the nonlasered group showed a tendency toward a greater increase in axial keratometry (6.63 ± 2.17 D vs. 5.60 ± 3.79 D, P = 0.613) and elevation (18.6 ± 15.4 vs. 15.2 ± 5.5, P = 0.656) than the lasered group. Conclusions Excimer laser ablation may be feasible for thinning and reshaping of SMILE-derived lenticules before reimplantation or allogenic transplantation. However, controlled lenticule dehydration before ablation is necessary in order to allow stromal thinning.

Optimization of spark plasma sintered titania for potential application as a keratoprosthesis skirt

The manufacture of mechanically strong and biocompatible titania (TiO2 ) materials is of vital importance for their application as corneal implant skirts. This study was aimed at optimizing the selection of raw powder and sintering conditions for TiO2 ceramics. TiO2 compacts were synthesized from five raw powders, denoted as Altair, Inframat, Alfa, Materion, and Amperit, respectively, by spark plasma sintering using different sintering parameters. The XRD and Raman results confirmed that the anatase TiO2 phase in the Inframat powder had converted completely to rutile TiO2 phase after sintering at 900°C and above. The nanoindentation results indicated that among the five types of TiO2 samples sintered at 1100°C, the Inframat pellets possessed the highest Youngs modulus and hardness. Additionally, when Materion samples were employed to study the effects of SPS parameters, a higher sintering temperature in the range of 1100-1300°C decreased the mechanical properties of sintered pellets probably due to the generation of more structural defects. Culture of human corneal stromal fibroblasts on the sintered sample surfaces showed that comparably high cell viability and proliferation were observed on all TiO2 samples except Amperit compared to positive control. Furthermore, cells cultured on Inframat TiO2 sintered in the temperature range of 900-1300°C exhibited viability and formation of focal adhesion complex similar to those on control, and those prepared at 1100°C had significantly higher cell proliferation indices than control. In conclusion, Inframat TiO2 consolidated at 1100°C by SPS was the best formulation for the preparation of mechanically strong and biocompatible Keratoprosthesis skirt.