Composite core-and-skirt collagen hydrogels with differential degradation for corneal therapeutic applications.

UNLABELLED: Scarcity of donor tissue to treat corneal blindness and the need to deliver stem cells or pharmacologic agents to ensure corneal graft survival are major challenges. Here, new composite collagen-based hydrogels are developed as implants to restore corneal transparency while serving as a possible reservoir for cells and drugs. The composite hydrogels have a centrally transparent core and embedded peripheral skirt of adjustable transparency and degradability, with the skirt exhibiting faster degradation in vitro. Both core and skirt supported human epithelial cell populations in vitro and the skirt merged homogeneously with the core material to smoothly distribute a mechanical load in vitro. After in vivo transplantation in rabbit corneas over three months, composites maintained overall corneal shape and integrity, while skirt degradation could be tracked in vivo and non-invasively due to partial opacity. Skirt degradation was associated with partial collagen breakdown, thinning, and migration of host stromal cells and macrophages, while the central core maintained integrity and transparency as host cells migrated and nerves regenerated. IMPACT: This study indicates the feasibility of a collagen-based composite hydrogel to maintain corneal stability and transparency while providing a degradable peripheral reservoir for cell or substance release.

Platelet-Rich Plasma Prolongs Myofibroblast Accumulation in Corneal Stroma with Incisional Wound.

PURPOSE: The purpose of this study was to determine whether platelet-rich plasma (PRP) has an effect on corneal stromal cells in a rat model of wound healing following corneal incision. MATERIALS AND METHODS: The effect of PRP on corneal wound healing in vivo was investigated in a corneal incision wound model in rats. 40 rats were wounded by deep corneal incision, and treated with either topically administered PRP (20 rats) or sodium chloride (20 rats). At 4 h and 1, 3, and 5 days after incision, α-smooth muscle actin (α SMA), SMAD2 and SMAD3 expression and apoptosis in stromal cells were evaluated by immunohistochemistry, and IL-1ß mRNA expression was evaluated by real time PCR. RESULTS: PRP-treated corneas exhibited reduced stromal cell apoptosis at day 3 and day 5 (p = 0.038, and <0.001, respectively) relative to controls. Interleukin-1ß mRNA expression, however, was unchanged in PRP-treated corneas relative to controls. Topical PRP treatment resulted in a higher proportion of αSMA-positive myofibroblasts recruited to the wound site relative to control corneas. PRP did not affect activation of SMAD2 but activation of SMAD3 was significantly reduced at day 1 (p = 0.001) and dramatically increased at day 5 (p = 0.032). CONCLUSIONS: PRP treatment resulted in suppressed stromal cell apoptosis followed by SMAD3 activation and a greater proportion of myofibroblasts present at the wound site. Suppression of stromal cell apoptosis after corneal wounding by use of a growth factor-rich formulation may lead to myofibroblast accumulation by modulation of the TGF-ß pathway.

Enhanced regeneration of corneal tissue via a bioengineered collagen construct implanted by a nondisruptive surgical technique.

Severe shortage of donor corneas for transplantation, particularly in developing countries, has prompted the advancement of bioengineered tissue alternatives. Bioengineered corneas that can withstand transplantation while maintaining transparency and compatibility with host cells, and that are additionally amenable to standardized low-cost mass production are sought. In this study, a bioengineered porcine construct (BPC) was developed to function as a biodegradable scaffold to promote corneal stromal regeneration by host cells. Using high-purity medical-grade type I collagen, high 18% collagen content and optimized EDC-NHS cross-linker ratio, BPCs were fabricated into hydrogel corneal implants with over 90% transparency and four-fold increase in strength and stiffness compared with previous versions. Remarkably, optical transparency was achieved despite the absence of collagen fibril organization at the nanoscale. In vitro testing indicated that BPC supported confluent human epithelial and stromal-derived mesenchymal stem cell populations. With a novel femtosecond laser-assisted corneal surgical model in rabbits, cell-free BPCs were implanted in vivo in the corneal stroma of 10 rabbits over an 8-week period. In vivo, transparency of implanted corneas was maintained throughout the postoperative period, while healing occurred rapidly without inflammation and without the use of postoperative steroids. BPC implants had a 100% retention rate at 8 weeks, when host stromal cells began to migrate into implants. Direct histochemical evidence of stromal tissue regeneration was observed by means of migrated host cells producing new collagen from within the implants. This study indicates that a cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate regenerative healing of the corneal stroma, and is compatible with human stem or organ-specific cells for future therapeutic applications as a stromal replacement for treating blinding disorders of the cornea.

Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model.

Inflammatory angiogenesis is the pathogenic mechanism of various sight-threatening eye diseases, among them corneal neovascularization. Current treatment options include steroids which have undesirable side effects, or anti-VEGF which has only limited efficacy. In an inflammatory environment, however, angiogenesis can be stimulated by numerous factors not directly targeted by anti-VEGF therapy. The aim of this study was to induce corneal inflammation leading to angiogenesis, and investigate the early, differential effects of steroid and anti-VEGF therapy at the cellular, tissue, and gene expression levels. Fifty-two Wistar rats received a single intrastromal corneal suture to induce a controlled inflammatory angiogenic response. Rats were subsequently treated with dexamethasone, rat specific anti-VEGF, or goat IgG (control), topically 4 times daily for 7 days. In vivo confocal microscopy of the cornea was performed longitudinally from 5 h up to 7 d to investigate morphology at the cellular and tissue-level. In vivo photographic vessel analysis and immunohistochemistry were also performed. RT-PCR for VEGF-A, FGF-2, IL-6, TNF-α, CXCL2, CCL2, CCL3 and DLL4 was performed at 24 h, and for VEGF-A, IL-6, TNF-α, FGF-2, CXCL2, CCL2, and CCL3 at 7 days. Early infiltration of CD11b + myeloid cells into the cornea at 5 h post-suture was delayed by both treatments relative to controls; however neither treatment was able to suppress accumulation of myeloid cells at day 2 or 7. Limbal vessel dilation was inhibited at 5 h by both treatments, but only dexamethasone showed sustained effect until day 2. Early macrophage recruitment was also suppressed by dexamethasone (but not by anti-VEGF) until day 2. Dexamethasone furthermore suppressed corneal neovascularization at day 7 by over 90%, whereas suppression by anti-VEGF was 14%. Despite differential suppression of vessel dilation, macrophage recruitment, and vascular invasion, anti-VEGF and dexamethasone both down-regulated VEGF-A and IL-6 expression at 24 h with sustained effect to 7 d. They also both down regulated FGF-2 and TNF-α at 24 h and CCL2 at 7 d. In conclusion, anti-angiogenic treatments influence early, pre-angiogenic tissue activity such as limbal vessel dilation, inflammatory cell infiltration of the stroma, and macrophage recruitment. Importantly, the differential effects of steroids and anti-VEGF treatment in suppressing neovascular growth could not be attributed to differential inhibition of several major angiogenic and inflammatory factors in the early pre-sprouting phase, including IL-6, VEGF-A, FGF-2, TNF-α, CCL2, CCL3, CXCL2, or DLL4.

An accurate method to determine Bowman's layer thickness in vivo in the human cornea.

PURPOSE: To determine an accurate value for Bowman's layer (BL) thickness in vivo in humans. METHODS: Seventeen corneal transplant patients were examined preoperatively by laser-scanning in vivo confocal microscopy (IVCM), and corneal buttons were removed postoperatively and sectioned for light microscopy (LM). Nine corneas with uniformly thick BL by LM were used for thickness measurement. In the uniformly thick samples, probable overestimation of BL thickness in vivo by a first in vivo method (Method 1) led to the development of a revised in vivo method (Method 2). Method 2 was used to measure BL thickness in 20 healthy volunteers. RESULTS: In nine patients, mean BL thickness prior to transplantation was 13.7 ± 1.6 µm by IVCM (Method 1) while BL thickness of the removed corneal button was 9.7 ± 1.7 µm by LM (P < 0.001). The correlation of BL thickness between IVCM (Method 1) and LM was poor (P = 0.226). In 20 right eyes of 20 normal corneas, both in vivo methods were used to determine BL thickness. Mean BL thickness by Method 1 was 13.2 ± 1.6 µm and by Method 2 was 9.1 ± 1.4 µm (P < 0.001). BL thickness measurements by both in vivo methods were highly correlated (P < 0.001). CONCLUSION: BL thickness by a revised in vivo method was close to LM values in this study and to values reported in fixed tissue in other studies. The authors believe this revised method provides the most accurate estimates of BL thickness in vivo to date.

The expression pattern of the subunit of chaperonin containing T-complex polypeptide 1 and its substrate, alpha-smooth muscle actin, during corneal wound healing.

PURPOSE: This study was designed to demonstrate the expression of the chaperonin containing T-complex polypeptide 1 (CCT) and alpha-smooth muscle actin (alpha-SMA), in normal corneas and corneas treated with ultraviolet radiation (UVR). The wound model chosen is previously characterized, the injury is mild and the cornea heals to transparency. METHODS: Rabbit corneas were exposed to UVR at the dose producing keratitis. The corneas were allowed to heal for up to 5 days and the paraffin-embedded tissue specimens were double stained and examined morphologically and immunohistochemically. Expression of CCT and alpha-SMA genes was investigated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: There was a front of repopulating keratocytes that showed positive staining for alpha-SMA after 3 days. The alpha-SMA mRNA was already strongly expressed after 1 day, whereas the expression level of CCT was increased after 2 days. After 5 days the levels were decreased. By this time the stroma was partly repopulated by keratocytes. CONCLUSION: In a mild wound, the expression of alpha-SMA mRNA is followed by expression of mRNA of at least one subunit of the complex folding alpha-SMA. At protein level, alpha-SMA is detected in the front line of repopulating keratocytes. Expression levels for both mRNAs decline as the stroma repopulation process progresses.

Biglycan gene expression in UVR-exposed rabbit corneas.

PURPOSE: It is known that stromal proteoglycans play an important role in the hydration and transparency of the mammalian cornea. Proteoglycans have been described as a pathological deposit in climatic proteoglycan stromal keratopathy, which is associated with chronic ultraviolet radiation (UVR) exposure. The expression of dermatan sulfate proteoglycan biglycan in the cornea was thus studied after exposure of rabbit eyes to UVR. METHODS: New Zealand albino rabbit corneas were exposed to UVR at 310 nm at the dose producing biomicroscopically significant keratitis (0.47 J/cm2). Animals were killed 3, 7 and 28 days after exposure (five rabbits in each group). Five rabbits were used as controls and did not receive any UVR treatment. Expression of biglycan mRNA in the corneas was investigated by competitive reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: There was no expression of biglycan mRNA in the control group. In the UVR-exposed groups, biglycan mRNA had still not been expressed 3 days after exposure. The expression of biglycan mRNA was observed in all UVR-treated corneas 7 days after exposure (p < 0.05). By 28 days after UVR exposure the expression of biglycan mRNA had decreased (not statistically significant). CONCLUSIONS: There is no detectable biglycan gene expression in the normal rabbit cornea. Ultraviolet radiation exposure leads to a distinct expression of biglycan mRNA in the rabbit cornea that decreases 4 weeks after exposure, indicating the involvement of biglycan in the corneal repair process. Biglycan appears to be a novel marker of corneal wound healing.