Depth-dependent mechanical properties of the human cornea by uniaxial extension.

The purpose of this study was to investigate the depth-dependent biomechanical properties of the human corneal stroma under uniaxial tensile loading. Human stroma samples were obtained after the removal of Descemet's membrane in the course of Descemet's membrane endothelial keratoplasty (DMEK) transplantation. Uniaxial tensile tests were performed at three different depths: anterior, central, and posterior on 2 × 6 × 0.15 mm strips taken from the central DMEK graft. The measured force-displacement data were used to calculate stress-strain curves and to derive the tangent modulus. The study showed that mechanical strength decreased significantly with depth. The anterior cornea appeared to be the stiffest, with a stiffness approximately 18% higher than that of the central cornea and approximately 38% higher than that of the posterior layer. Larger variations in mechanical response were observed in the posterior group, probably due to the higher degree of alignment of the collagen fibers in the posterior sections of the cornea. This study contributes to a better understanding of the biomechanical tensile properties of the cornea, which has important implications for the development of new treatment strategies for corneal diseases. Accurate quantification of tensile strength as a function of depth is critical information that is lacking in human corneal biomechanics to develop numerical models and new treatment methods.

Comparison of the Effects of Temperature and Dehydration Mode on Glycerin-Based Approaches to SMILE-Derived Lenticule Preservation.

PURPOSE: The aim of this study was to explore the optimal method of small-incision lenticule extraction (SMILE)-derived lenticules, subjected to long-term preservation using glycerol, under a range of temperatures, and using an array of dehydration agents. METHODS: In total, 108 myopic lenticules were collected from patients undergoing the SMILE procedure. Fresh lenticules served as a control group for this study, whereas all other lenticules were separated into 8 groups, which were preserved at 4 different temperatures (room temperature [RT], 4, -20, and -80°C) with or without silica gel in anhydrous glycerol. Evaluated parameters included thickness, transmittance, hematoxylin and eosin staining, transmission electron microscopy, and immunohistochemistry analyses. RESULTS: After a 3-month preservation period, lenticular thickness in these different groups was significantly increased, particularly for samples stored at RT. The mean percentage transmittance of lenticules stored at -80°C with or without silica gel was closest to that of fresh lenticules. Hematoxylin and eosin staining revealed sparsely arranged collagen fibers that were more scattered in preserved lenticules relative to fresh lenticules, particularly in RT samples. Transmission electron microscopy revealed that the fibril bundles densities in lenticules stored at RT were significantly less than those stored at other temperatures. Immunohistochemistry analyses revealed reductions in or loss of CD45 and human leukocyte antigens in all preserved lenticules relative to control samples. CONCLUSIONS: Of the tested approaches, the preservation of SMILE-derived lenticules over a 3-month period was optimal at -80°C with or without silica gel in anhydrous glycerol.

Collagen XIV Is an Intrinsic Regulator of Corneal Stromal Structure and Function.

Collagen XIV is poorly characterized in the body, and the current knowledge of its function in the cornea is limited. The aim of the current study was to elucidate the role(s) of collagen XIV in regulating corneal stromal structure and function. Analysis of collagen XIV expression, temporal and spatial, was performed at different postnatal days (Ps) in wild-type C57BL/6 mouse corneal stromas and after injury. Conventional collagen XIV null mice were used to inquire the roles that collagen XIV plays in fibrillogenesis, fibril packing, and tissue mechanics. Fibril assembly and packing as well as stromal organization were evaluated using transmission electron microscopy and second harmonic generation microscopy. Atomic force microscopy was used to assess stromal stiffness. Col14a1 mRNA expression was present at P4 to P10 and decreased at P30. No immunoreactivity was noted at P150. Abnormal collagen fibril assembly with a shift toward larger-diameter fibrils and increased interfibrillar spacing in the absence of collagen XIV was found. Second harmonic generation microscopy showed impaired fibrillogenesis in the collagen XIV null stroma. Mechanical testing suggested that collagen XIV confers stiffness to stromal tissue. Expression of collagen XIV is up-regulated following injury. This study indicates that collagen XIV plays a regulatory role in corneal development and in the function of the adult cornea. The expression of collagen XIV is recapitulated during wound healing.

Tissue engineering of corneal stroma via melt electrowriting.

The cornea serves as the main refractive component of the eye with the corneal stroma constituting the thickest component in a stratified layered system of epithelia, stroma, and endothelium. Current treatment options for patients suffering from corneal diseases are limited to transplantation of a human donor cornea (keratoplasty) or to implantation of an artificial cornea (keratoprosthesis). Nevertheless, donor shortage and failure of artificial corneas to integrate with local tissue constitute important problems that have not been yet circumvented. Recent advances in biofabrication have made great progress toward the manufacture of tailored biomaterial templates with the potential of guiding partially or totally the regeneration process of the native cornea. However, the role of the corneal stroma on current tissue engineering strategies is often neglected. Here, we achieved a tissue-engineered corneal stroma substitute culturing primary keratocytes on scaffolds prepared via melt electrowriting (MEW). Scaffolds were designed to contain highly organized micrometric fibers to ensure transparency and encourage primary human keratocytes to self-orchestrate their own extracellular matrix deposition and remodeling. Results demonstrated reliable cell attachment and growth over a period of 5 weeks and confirmed the formation of a dense and highly organized de novo tissue containing collagen I, V, and VI as well as Keratocan, which resembled very closely the native corneal stoma. In summary, MEW brings us closer to the biofabrication of a viable corneal stroma substitute.

Corneal stromal roughness after VisuMax and Intralase femtosecond laser photodisruption: An atomic force microscopy study.

PURPOSE: To compare the induced corneal stromal bed roughness measured with atomic force microscopy (AFM) after LASIK flap creation with the IntraLase 60 kHz and the VisuMax femtosecond laser platforms. METHODS: Three freshly enucleated porcine eyes were operated with each femtosecond laser in this experimental study. Standard LASIK treatment parameters were used for the experiment. After LASIK flap creation, the corneal stromal roughness was assessed using a JPK NanoWizard II® AFM in contact mode immersed in liquid. Olympus OMCL-RC800PSA commercial silicon nitride cantilever tips were used. Surface measurements were made in 10 regions of the central cornea of each sample measuring 20 x 20 microns, at 512 x 512 point resolution. Roughness was measured using the root-mean-square (RMS) value within the given regions. RESULTS: Measurements from 30 regions of the 3 eyes (10 measurements per eye) in the Intralase (FS1) group, and 30 regions of the 3 eyes (10 measurements per eye) in the VisuMax (FS2) group were analyzed. There was a statistically significant difference in mean ± standard deviation RMS values between the FS1 and the FS2 groups (360 ± 120 versus 230 ± 100 nm respectively; P< 0.00001). CONCLUSION: This AFM study indicates that the surface of the stromal bed after LASIK flap creation is smoother in the FS2 group than the FS1 group.

Corneal Stromal Regeneration Therapy for Advanced Keratoconus: Long-term Outcomes at 3 Years.

PURPOSE: To report the 3-year clinical outcomes of corneal stromal cell therapy consisting of the intrastromal implantation with autologous adipose-derived adult stem cells (ADASCs), and decellularized or ADASC-recellularized human donor corneal laminas in advanced keratoconus. METHODS: Fourteen patients were enrolled in 3 experimental groups. Group 1 (G-1) patients underwent implantation of ADASCs alone (3 × 106 cells/1 mL) (n = 5). Group 2 (G-2) patients received a 120-µm decellularized corneal stroma lamina (n = 5). Group 3 (G-3) patients received a 120-µm lamina recellularized with ADASCs (1 × 106 cells/1 mL) (n = 4). ADASCs were obtained by elective liposuction. Implantation was performed into a femtosecond pocket under topical anesthesia. RESULTS: At 3 years, a significant improvement of 1 to 2 logMAR lines in uncorrected distance visual acuity was observed in all groups. A statistically significant decrease in corrected distance visual acuity was obtained in G-2 and G-3 (P < 0.001) when compared with that of G-1. Rigid contact lens distance visual acuity showed a statistically significant worsening in G-2 (P < 0.001) compared with that of G-1. A statistically significant increase in central corneal thickness was observed in G-2 (P = 0.012) and G-3 (P < 0.001); in the Scheimpflug corneal topography, the thinnest point was observed in G-2 (P = 0.007) and G-3 (P = 0.001) when compared with that of G-1. CONCLUSIONS: Intrastromal implantation of ADASCs and decellularized or ADASC-recellularized human corneal stroma laminas did not have complications at 3 years. The technique showed a moderate improvement in (uncorrected distance visual acuity) and (corrected distance visual acuity) in advanced keratoconus.

Corneal stroma regeneration: Preclinical studies.

Corneal grafting is one of the most common and successful forms of human tissue transplantation in the world, but the need for corneal grafting is growing and availability of human corneal donor tissue to fulfill this increasing demand is not assured worldwide. The stroma is responsible for many features of the cornea, including its strength, refractive power and transparency, so enormous efforts have been put into replicating the corneal stroma in the laboratory to find an alternative to classical corneal transplantation. Unfortunately this has not been yet accomplished due to the extreme difficulty in mimicking the highly complex ultrastructure of the corneal stroma, and none of the obtained substitutes that have been assayed has been able to replicate this complexity yet. In general, they can neither match the mechanical properties nor recreate the local nanoscale organization and thus the transparency and optical properties of a normal cornea. In this context, there is an increasing interest in cellular therapy of the corneal stroma using Induced Pluripotent Stem Cells (iPSCs) or mesenchymal stem cells (MSCs) from either ocular or extraocular sources, as they have proven to be capable of producing new collagen within the host stroma, modulate preexisting scars and enhance transparency by corneal stroma remodeling. Despite some early clinical data is already available, in the current article we will summary the available preclinical evidence about the topic corneal stroma regeneration. Both, in vitro and in vivo experiments in the animal model will be shown.

Decellularized human corneal stromal cell sheet as a novel matrix for ocular surface reconstruction.

The shortage of donor corneas as well as the limitations of tissue substitutes leads to the necessity to develop alternative materials for ocular surface reconstruction. Corneal surface substitutes must fulfill specific requirements such as high transparency, low immunogenicity, and mechanical stability combined with elasticity. This in vitro study evaluates a decellularized matrix secreted from human corneal fibroblasts (HCF) as an alternative material for ocular surface reconstruction. HCF from human donors were cultivated with the supplementation of vitamin C to form a stable and thick matrix. Furthermore, due to enhanced cultivation time, a three-dimensional like multilayered construct which partly mimics the complex structure of the corneal stroma could be generated. The formed human cell-based matrices (so-called cell sheets [CS]) were subsequently decellularized. The complete cell removal, collagen content, ultrastructure, and cell toxicity of the decellularized CS (DCS) as well as biomechanical properties were analyzed. Surgical feasibility was tested on enucleated porcine eyes. After decellularization and sterilization, a transparent, thick, cell free, and sterile tissue substitute resulted, which allowed expansion of limbal epithelial stem cells with no signs of cytotoxicity, and good surgical feasibility. DCS seem to be a promising new corneal tissue substitute derived from human cells without the limitation of donor material; however, future in vivo studies are necessary to further elucidate its potential for ocular surface reconstruction.

Collagen XII Is a Regulator of Corneal Stroma Structure and Function.

Purpose: The aim of this study was to determine the roles of collagen XII in the regulation of stromal hierarchical organization, keratocyte organization, and corneal mechanics. Methods: The temporal and spatial expression of collagen XII at postnatal days 4, 10, 30, 90, and 150 were evaluated in wild-type (WT) mice. The role of collagen XII in hierarchical organization was analyzed by measuring fibril diameter and density, as well as stromal lamellar structure, within ultrastructural micrographs obtained from WT and collagen XII-deficient mice (Col12a1-/-). Keratocyte morphology and networks were assessed using actin staining with phalloidin and in vivo confocal microscopy. The effects of collagen XII on corneal biomechanics were evaluated with atomic force microscopy. Results: Collagen XII was localized homogeneously in the stroma from postnatal day 4 to day 150, and protein accumulation was shown to increase during this period using semiquantitative immunoblots. Higher fibril density (P < 0.001) and disruption of lamellar organization were found in the collagen XII null mice stroma when compared to WT mice. Keratocyte networks and organization were altered in the absence of collagen XII, as demonstrated using fluorescent microscopy after phalloidin staining and in vivo confocal microscopy. Corneal stiffness was increased in the absence of collagen XII. Young's modulus was 16.2 ± 5.6 kPa in WT and 32.8 ± 6.4 kPa in Col12a1-/- corneas. The difference between these two groups was significant (P < 0.001, t-test). Conclusions: Collagen XII plays a major role in establishing and maintaining stromal structure and function. In the absence of collagen XII, the corneal stroma showed significant abnormalities, including decreased interfibrillar space, disrupted lamellar organization, abnormal keratocyte organization, and increased corneal stiffness.

Human allograft refractive lenticular implantation for high hyperopiccorrection.

Hyperopia is a common form of refractive error in the United States. Many refractive errors can be treated with refractive surgery methods such as laser in-situ keratomileusis and photorefractive keratectomy; however, in patients with large degrees of hyperopia (≥+5.0 diopters [D]), these surgical methods are limited because of higher rates of refractive regression. Lenticule Intrastromal Keratoplasty (LIKE) is a surgical procedure that can be used to correct refractive errors in patients with high hyperopia. The authors describe the first intrastromal implantation of an allograft lenticule performed for the primary correction of hyperopia in the United States, and demonstrate that LIKE is potentially an effective procedure for the correction of high hyperopia. Mechanisms for achieving the intended refractive correction and the complications our patient experienced, including epithelial ingrowth and flap necrosis, are discussed.

Fiber reinforced GelMA hydrogel to induce the regeneration of corneal stroma.

Regeneration of corneal stroma has always been a challenge due to its sophisticated structure and keratocyte-fibroblast transformation. In this study, we fabricate grid poly (ε-caprolactone)-poly (ethylene glycol) microfibrous scaffold and infuse the scaffold with gelatin methacrylate (GelMA) hydrogel to obtain a 3 D fiber hydrogel construct; the fiber spacing is adjusted to fabricate optimal construct that simulates the stromal structure with properties most similar to the native cornea. The topological structure (3 D fiber hydrogel, 3 D GelMA hydrogel, and 2 D culture dish) and chemical factors (serum, ascorbic acid, insulin, and ß-FGF) are examined to study their effects on the differentiation of limbal stromal stem cells to keratocytes or fibroblasts and the phenotype maintenance, in vitro and in vivo tissue regeneration. The results demonstrate that fiber hydrogel and serum-free media synergize to provide an optimal environment for the maintenance of keratocyte phenotype and the regeneration of damaged corneal stroma.

Accelerated, Pulsed Collagen Cross-Linking versus the Dresden Protocol in Keratoconus: A Case Series.

PURPOSE: The aim of our study was to compare the depth of the demarcation line developing in the cornea after the standard Dresden protocol versus the accelerated, pulsed, epithelium-off corneal collagen cross-linking (CXL). METHODS: This was a nonrandomized, retrospective case series. Patients with progressive keratoconus were treated with either the standard Dresden protocol (Group 1) or accelerated, epithelium-off CXL using the Avedro (Waltham, MA, USA) device (Group 2). The accelerated CXL protocol involved 18 min of pulsed ultraviolet-A (20 mW/cm2, 7.2 J/cm2, pulsed pro-file: 1 s on, 2 s off). The depth of the demarcation line was measured about 3 months postoperatively. RESULTS: Fifty-nine eyes of 35 subjects were included in the analysis. Group 1 consisted of 19 eyes, and Group 2 of 40 eyes. The mean age of the participants was 22.21 years in Group 1 and 26.55 years in Group 2 (p = 0.184). The mean preoperative K value was 44.89 D in Group 1 and 45.20 D in Group 2 (p = 0.768). The depth of the demarcation line was 322.50 µm in Group 1 and 319.95 µm in Group 2 (p = 0.937). CONCLUSIONS: The demarcation line depth was not statistically significantly different between the two protocols. The significance of the demarcation line depth has not been fully clarified in the literature. Our results support the contention that these two techniques may have similar structural outcomes and ef-ficacies in the treatment of keratoconus.

Banking of corneal stromal lenticules: a risk-analysis assessment with the EuroGTP II interactive tool.

We evaluated the feasibility and performed a risk-benefit analysis of the storage and widespread distribution of stromal lenticules for clinical application using a new systematic tool (European Good Tissue and cells Practices II-EuroGTP II tool), specifically designed for assessing the risk, safety and efficacy of substances of human origin. Three types of potential tissue preparations for human stromal lenticules were evaluated: cryopreserved, dehydrated and decellularized. The tool helps to identify an overall risk score (0-2: negligible; 2-6: low; 6-22: moderate; > 22: high) and suggests risk reduction strategies. For all the three types of products, we found the level of risk to be as "moderate". A process validation, pre-clinical in vitro and in vivo evaluations and a clinical study limited to a restricted number of patients should therefore be performed in order to mitigate the risks. Our study allowed to establish critical points and steps necessary to implement a new process for safe stromal lenticule preparation by the eye banks to be used in additive keratoplasty. Moreover, it shows that the EuroGTP II tool is useful to assess and identify risk reduction strategies for introduction of new Tissue and Cellular Therapies and Products into the clinical practice.

Healing characteristics of acellular porcine corneal stroma following therapeutic keratoplasty.

BACKGROUND: Acellular porcine corneal stroma (APCS) has proven to be a promising alternative to traditional corneal grafts. This prospective case series was conducted to further investigate the healing characteristics of APCS following keratoplasty. METHODS: Twenty-seven patients undergoing APCS implantation to treat infectious keratitis were included. The patients were followed up for 12 months after surgery. The main outcome measures included visual acuity, corneal transparency, graft thickness, and cellular and nerve regeneration. RESULTS: In the operated eyes, the best-corrected visual acuity (BCVA, in logarithm of the minimal angle of resolution [logMAR] units) increased from 1.23 ± 0.95 logMAR before surgery to 0.23 ± 0.18 logMAR at 12 months after surgery (P < .001). The contrast sensitivity was still evidently reduced, especially at higher spatial frequencies. Gradual transparency improvement was observed in APCS grafts post-operatively. After implantation, the APCS graft thickness initially increased (day 1 = 592.41 ± 52.69 µm) but then continuously decreased until 3 months after surgery (1 month = 449.26 ± 50.38 µm; 3 months = 359.63 ± 34.14 µm, P < .001). Graft reepithelialization was completed within 1 week. In the in vivo confocal microscopy scans, host keratocytes began to repopulate the APCS grafts between 3 and 6 months post-operatively; subbasal nerve regeneration was only noted in 18.52% (5/27) of the eyes by 12 months after surgery. CONCLUSIONS: Acellular porcine corneal stroma functions as an effective alternative to human corneal tissue in lamellar keratoplasty. However, APCS is somewhat different from fresh human cornea in term of the post-operative healing process, which warrants the attention of both clinicians and patients.

Cell Loaded GelMA:HEMA IPN hydrogels for corneal stroma engineering.

Stroma is the main refractive element of the cornea and damage to it is one of the main causes of blindness. In this study, cell loaded hydrogels of methacrylated gelatin (GelMA) and poly(2-hydroxyethyl methacrylate) (pHEMA) (8:2) interpenetrating network (IPN) hydrogels were prepared as the corneal stroma substitute and tested in situ and in vitro. Compressive modulus of the GelMA hydrogels was significantly enhanced with the addition of pHEMA in the structure (6.53 vs 155.49 kPa, respectively). More than 90% of the stromal keratocytes were viable in the GelMA and GelMA-HEMA hydrogels as calculated by Live-Dead Assay and NIH Image-J program. Cells synthesized representative collagens and proteoglycans in the hydrogels indicating that they preserved their keratocyte functions. Transparency of the cell loaded GelMA-HEMA hydrogels was increased significantly up to 90% at 700 nm during three weeks of incubation and was comparable with the transparency of native cornea. Cell loaded GelMA-HEMA corneal stroma model is novel and reported for the first time in the literature in terms of introduction of cells during the preparation phase of the hydrogels. The appropriate mechanical strength and high transparency of the cell loaded constructs indicates a viable alternative to the current devices used in the treatment of corneal blindness.

Collagen fibrils and proteoglycans of peripheral and central stroma of the keratoconus cornea - Ultrastructure and 3D transmission electron tomography.

Keratoconus (KC) is a progressive corneal disorder in which vision gradually deteriorates as a result of continuous conical protrusion and the consequent altered corneal curvature. While the majority of the literature focus on assessing the center of this diseased cornea, there is growing evidence of peripheral involvement in the disease process. Thus, we investigated the organization of collagen fibrils (CFs) and proteoglycans (PGs) in the periphery and center of KC corneal stroma. Three-dimensional transmission electron tomography on four KC corneas showed the degeneration of microfibrils within the CFs and disturbance in the attachment of the PGs. Within the KC corneas, the mean CF diameter of the central-anterior stroma was significantly (p ˂ 0.001) larger than the peripheral-anterior stroma. The interfibrillar distance of CF was significantly (p ˂ 0.001) smaller in the central stroma than in the peripheral stroma. PGs area and the density in the central KC stroma were larger than those in the peripheral stroma. Results of the current study revealed that in the pre- Descemet's membrane stroma of the periphery, the degenerated CFs and PGs constitute biomechanically weak lamellae which are prone to disorganization and this suggests that the peripheral stroma plays an important role in the pathogenicity of the KC cornea.

Tissue adhesive hyaluronic acid hydrogels for sutureless stem cell delivery and regeneration of corneal epithelium and stroma.

Regeneration of a severely damaged cornea necessitates the delivery of both epithelium-renewing limbal epithelial stem cells (LESCs) and stroma-repairing cells, such as human adipose-derived stem cells (hASCs). Currently, limited strategies exist for the delivery of these therapeutic cells with tissue-like cellular organization. With the added risks related to suturing of corneal implants, there is a pressing need to develop new tissue adhesive biomaterials for corneal regeneration. To address these issues, we grafted dopamine moieties into hydrazone-crosslinked hyaluronic acid (HA-DOPA) hydrogels to impart tissue adhesive properties and facilitate covalent surface modification of the gels with basement membrane proteins or peptides. We achieved tissue-like cellular compartmentalization in the implants by encapsulating hASCs inside the hydrogels, with subsequent conjugation of thiolated collagen IV or laminin peptides and LESC seeding on the hydrogel surface. The encapsulated hASCs in HA-DOPA gels exhibited good proliferation and cell elongation, while the LESCs expressed typical limbal epithelial progenitor markers. Importantly, the compartmentalized HA-DOPA implants displayed excellent tissue adhesion upon implantation in a porcine corneal organ culture model. These results encourage sutureless implantation of functional stem cells as the next generation of corneal regeneration.

Adhesion Strength and Rolling Properties of Descemet Membrane Endothelial Keratoplasty Grafts in a Rabbit Eye Model.

Purpose: To investigate the optimal time for Descemet membrane endothelial keratoplasty (DMEK) graft peeling, and to analyze the rolling properties of endothelial denuded grafts in a rabbit eye model. Materials and Methods: The vertical peeling force required to peel 1 mm wide Descemet membrane (DM) strips, was measured as the change in weight of the system during force application in a rabbit model. Twenty-one rabbit corneoscleral rims were stored in phosphate-buffered saline (PBS) at 4°C, and force analysis was performed at days 1, 5, or 21 after harvesting. After half of the strips of day 5 corneas were peeled and analyzed, the rims were moved to Optisol GS at 4°C, and the remaining strips were peeled off for force analysis at day 10. Separate DM grafts (n = 7) were analyzed by intraoperative optical coherence tomography (OCT) to determine the tissue rolling diameter before and after removal of endothelial cells by a swab. Unpaired t-test was used for statistical analysis. Results: There was a decrease in DM peeling force (p = .008) between days 1 and 5 (556.04 ± 111.76 and 324.30 ± 96.4 mg, respectively), and no difference between days 5 and 21 (p = .53). Peeling force for day 5 corneas placed in Optisol was higher at day 10 (324.30 ± 96.4 to 669.92 ± 166.24 mg, p = .005). The average rolling diameter of DM grafts was similar before and after the removal of endothelial cells (257.9 ± 131.1 and 249.8 ± 126.6 µm, respectively). Conclusions: DMEK Graft procurement could be potentially facilitated by lower DM-stromal adhesion strength at day five after obtaining corneoscleral rims, in a rabbit eye model. Time in the storage medium may influence adhesion strength. Endothelial cells do not appear to play a significant role in the rolling diameter of DM grafts.

Shear-induced alignment of collagen fibrils using 3D cell printing for corneal stroma tissue engineering.

The microenvironments of tissues or organs are complex architectures comprised of structural proteins including collagen. Particularly, the cornea is organized in a lattice pattern of collagen fibrils which play a significant role in its transparency. This paper introduces a transparent bioengineered corneal structure for transplantation. The structure is fabricated by inducing shear stress to a corneal stroma-derived decellularized extracellular matrix bioink based on a 3D cell printing technique. The printed structure recapitulates the native macrostructure of the cornea with aligned collagen fibrils which results in the construction of a highly matured and transparent cornea stroma analog. The level of shear stress, controlled by the various size of the printing nozzle, manipulates the arrangement of the fibrillar structure. With proper parameter selection, the printed cornea exhibits high cellular alignment capability, indicating a tissue-specific structural organization of collagen fibrils. In addition, this structural regulation enhances critical cellular events in the assembly of collagen over time. Interestingly, the collagen fibrils that remodeled along with the printing path create a lattice pattern similar to the structure of native human cornea after 4 weeks in vivo. Taken together, these results establish the possibilities and versatility of fabricating aligned collagen fibrils; this represents significant advances in corneal tissue engineering.

Biobanking of Dehydrated Human Donor Corneal Stroma to Increase the Supply of Anterior Lamellar Grafts.

PURPOSE: To investigate the effect of dehydration on human donor corneal stroma for biobanking. METHODS: Epithelium and endothelium of research-grade human donor corneas (n = 12) were scraped off, leaving a bare stroma with attached sclera. The tissues were placed in a large Petri dish prefilled with silica gel in the periphery and stored at room temperature for 14 days. At the end of preservation, the tissues were rehydrated by being submerged in phosphate-buffered saline for 15 minutes. Transparency (using a custom-built device) and thickness (using optical coherence tomography) measurements were recorded before dehydration, after dehydration, and after rehydration of the tissues. Periodic acid-Schiff and alpha-smooth muscle actin (α-SMA) staining before dehydration and after rehydration were performed to determine the presence of keratocytes and expression of α-SMA. Tensile stress-strain before dehydration and after rehydration was performed to evaluate the biomechanical properties. RESULTS: No difference in corneal transparency before dehydration (69.57 ± 6.41%) and after rehydration (67.37 ± 2.82%), P = 0.36, was observed. The corneas were more compact after dehydration. A significant change in thickness between before dehydration (625.8 ± 75.58 µm) and after rehydration (563.6 ± 15.77 µm) stage, P = 0.03, was noticed. The thickness was reduced to 147.6 ± 3.71 µm when dehydrated. Periodic acid-Schiff staining showed presence of stromal keratocytes and α-SMA protein expressed in control, dehydrated, and rehydrated corneas. There was no significant difference in the stiffness between control (27.86 ± 11.65 MPa) and rehydrated corneas (31.46 ± 11.41 MPa). CONCLUSIONS: Human donor corneal stroma can be biobanked for up to 2 weeks in a dehydrated condition without losing their molecular or biomechanical properties after rehydration.