Corneal Stroma Regeneration with Collagen-Based Hydrogel as an Artificial Stroma Equivalent: A Comprehensive In Vivo Study

Corneal transplantation is the widely accepted treatment to restore sight for corneal blindness. To date, because of the global donor cornea shortage, there is a need for alternatives to human donor corneas. Biocompatible collagen is an excellent candidate material for corneal repair in the view of biomimetics. Herein a class of polyrotaxane multiple aldehyde (PRA) cross-linkers based on the host-guest supramolecules of α-cyclodextrins and poly(ethylene glycol) is prepared to cross-link with collagen to fabricate materials for corneal repair. Aldehyde groups from rotaxanes and α-cyclodextrin units can synergistically improve the mechanical and optical properties of PRA cross-linked collagen membranes (Col-PRAs). Compared with counterparts cross-linked by traditional a cross-linker of 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide and N-hydroxy-succinimide, Col-PRAs have better mechanical properties, especially suture resistance as well as optical properties. In vivo lamellar keratoplasty results indicate that Col-PRAs not only can bear tight suturing on a rabbit cornea but also are prone to the remodeling of the epithelium and stroma of the cornea due to the outstanding cell adhesion and proliferation. These novel Col-PRAs exhibit great potential for use in the corneal regeneration.

Stable and biocompatible fibrillar hydrogels based on the self-crosslinking between collagen and oxidized chondroitin sulfate

The three-dimensional (3D) microanatomy of the cornea is the major determinant of its optical and mechanical properties. Scanning electron microscopy (SEM) is the most commonly used method to obtain information on the overall 3D microanatomy of organs. However, SEM has not been successful in revealing the 3D microanatomy of the cornea, because the interior of the cornea is too compact to be explored by the electron beam. In this study, the 3D organisation of the cells and extracellular materials of human and rabbit corneas was examined after exposure by HCl and NaOH digestion, and by microdissection by the adhesive tape method. In the cornea of both species, all epithelial cells exhibited microplicae regardless of their location. This raises doubts about the tear film-holding role assigned to the microplicae of the superficial cells. Human and rabbit corneas differed in the collagen fibre patterns of the epithelial basement membranes. The 3D organisation of the stromal lamellae was similar in both species. In humans and rabbits, the keratocytes showed similar 3D features. However, the surface of human keratocytes located near Descemet's membrane exhibited small fenestrations that were not present in the rabbit keratocytes. The pattern of keratocyte innervation by the stromal neural plexus and 3D keratocyte microanatomy confirms that keratocytes form a large intercommunicating network within the corneal stroma. Two morphologically discrete subpopulations of keratocytes located at different stromal levels were identified in both human and rabbit corneas, suggesting that keratocytes are not functionally homogeneous. In addition, the density of the stromal neural plexus appeared to be greater in rabbits than in humans. Clear differences between human and rabbit corneas were observed in the collagen arrangement in Descemet's membrane, which may reflect their different biomechanical requirements.

To evaluate the efficacy of recombinant human nerve growth factor-loaded amniotic membrane (rhNGF-AM) on corneal epithelial and nerve regeneration in rabbit model. Freshly prepared human amniotic membrane (AM) were immersed into PBS buffer containing 100 or 500 µg/mL rhNGF for 15, 30, and 60min at 4°C. The in vitro release kinetics of rhNGF was measured with ELISA. For in vivo evaluation, the AM were immersed with 500 µg/mL rhNGF for 30min. Fifty-seven rabbits were selected to establish corneal epithelial defect model. In addition to the 19 rabbits in control group, 38 rabbits received AM transplantation with or without rhNGF after the removal of central epithelium. Corneal epithelial defect area, sub-epithelial nerve fiber density, corneal sensitivity, rhNGF contents in resident AM and corneas were measured after the surgery. rhNGF was sustained release from the AM within 14d in vitro, with the positive correlation with initial immersion concentration. The immersion of AM in 500 µg/mL rhNGF for 30min achieved the most stable release within 14d. After transplantation in rabbit cornea, a high concentration of rhNGF in resident rhNGF-AM and cornea was maintained within 8d. Corneal epithelial healing, nerve fiber regeneration and the recovery of corneal sensitivity were significantly accelerated after the rhNGF-AM transplantation when compared to simple AM transplantation (all P<0.05). Simple immersion of AM achieves the sustained release of rhNGF, and promotes corneal epithelial wound healing and nerve regeneration, as well as the recovery of corneal sensitivity in rabbit.

Corneal disease is the main cause of blindness and keratoplasty is the only widely accepted treatment. Shortage of donor tissue makes the biomaterials for corneal regeneration a hot area of research. Collagen is the main component of corneal stroma, so collagen becomes a promising material for corneal repair. However, due to the drawbacks of collagen, it needs to be further modified to satisfy the requirement of corneal regeneration. In this article, we highlight the importance of collagen materials for corneal repair, and summarize several methods of preparing collagen based corneal regeneration materials, including chemical crosslinking, plastic compression of collagen, and collagen vitrigel. These modification methods can make collagen membranes with remarkable properties such as enough mechanical and suture retention strength, antibacterial property and excellent optical property. These materials may provide potential treatment for corneal disease.

Corneal stromal filler injection of gelatin-based photocurable hydrogels for maintaining the corneal thickness and reconstruction of corneal stroma

Targeting Ca2+-dependent pathways to promote corneal epithelial wound healing induced by CISD2 deficiency

Objective Corneal defect is a common disease in ophthalmology caused by trauma, inflammation, drug toxicity, or surgery. To investigate the effect of germinal peptide eye drop on corneal epithelial and stromal defects after lamellar keratectomy in rabbit model. Methods Eighty-five male New Zealand white rabbits were divided into five groups: Germinal Peptide eye drop at three different concentration groups, normal saline (negative control group), recombinant human epidermal growth factor (rh-EGF) eye drop (positive control group). Corneal epithelial and stromal defects of around 150–200 μm in depth were created with an 8 mm diameter trephine in the center of the right eyes of all animals. Results Germinal peptide eye drop with the concentration of 0.001%, 0.002%, and 0.004% and rh-EGF eye drop were more effective in promoting healing, reducing opacity, and edema during the process of corneal epithelial and stromal defect regeneration compared with 0.9% normal saline. No significant difference was observed among the three different doses of germinal peptide eye drop. Compared with the saline control group, the structures of the regenerated corneas were more orderly and less inflammatory cell infiltration was observed in the germinal peptide eye drop groups and the rh-EGF eye drop group. Conclusion Germinal peptide eye drop (0.001%, 0.002%, and 0.004%) can significantly stimulate the regeneration of corneal epithelia and stroma and reduce corneal opacity and edema. Dose dependency was not observed in the current study.

To evaluate the potential use of decellularized porcine stromal matrix (PSM) for reconstruction of corneal stroma in a rabbit model. Ten chinchilla bastard rabbit corneas were exposed to a circular half-thickness keratotomy with a 3.0 mm diameter at the central cornea. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra-acetic acid (EDTA, 0.1%), aprotinin (10 K IU/ml) and 0.3% sodium dodecyl sulphate (SDS). The 3.0 mm in diameter decellularized corneal stromal xenograft was inserted into the pocket, and the incision was closed with four 10.0 nylon sutures. Clinical photographs were taken at day 1, day 7, day 30 and on a monthly basis for up to 6 months after transplantation. Six months after surgery, the rabbits were killed and eyes were enucleated. Haematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm the complete removal of the corneal cells after decellularization of porcine corneas and repopulation with rabbit cells. Alcian blue staining was performed for analysing the structure of the extracellular matrix (ECM). Efficient elimination of porcine cells was achieved by our decellularization protocol and confirmed via HE and DAPI stainings. Moreover, the major histoarchitectural ECM structure had been maintained as visualized by the alcian blue stain. Finally, the PSM was biocompatible with the host's epithelium evidenced as a regrowth covering the exposed xenograft. This novel technique of tissue engineering may provide one of many solutions to addressing anterior corneal pathological conditions in the face of a shortage of human corneal material.

In May 1933, some scabs, which had been obtained from the skin of a sheep suspected to suffer from sheep-pox were sent to our laboratory from X district. To the scabs. was added 1% carbol solution in 1: 4 proportion and, after shaking for 3 hours, the suspension was centrifugalized. The sediment was washed 3 or 4 times with saline solution and it was then emulsified in 1: 4 proportion with carbol (0.5%)-glycerin (60%) solution.Animal experiments were then performed with the scab emulsion thus produced and especially the vaccinization with this scabvirus was tried. The results obtained are summarized as follows: (1) Smearing of the scab-virus on the scarified cornea of sheep caused a clouding of the inoculated area on the 3rd day and this reaction continued 2 weeks. In pig, a slight clouding appeared on the 11th day after inoculation and it persisted for 40 days. In calves, a clouding of cornea occurred on the 3rd. to 4th day after inoculation and continued one month. We, however, couldn't observe such a reaction in goats, rabbits or guinea-pigs.(2) When the scab-virus was inoculated on the incised skin of sheep, vesicle appeared on the 3rd day and developed to its maximum on the 5th to 6th day. The desiccation stage comes generally after 2 weeks and the formed scabs begin to fall off after 3 weeks.(3) The dermal inoculation with sheep-passage virus as well as the intravenous injection with the 2nd passage-virus produced a beautiful, confluent eruption on the skin of sheep. If the blood, liver and spleen of thus infected sheep are inoculated on the skin of sheep or goats, these materials can also produce distinct eruption.(4) The scab-virus filtered through N or V candle of Berkefeld filter, produced a definite eruption by dermal or intradermal inoculation into sheep or goats. It was then proved that the virus under discussion is filterable.(5) When the scab-virus was inoculated on the skin of goats, the eruption developed its maximum nearly on the 6th day (Fig. 4) and the formed scabs began to fall off on about the 16th day.(6) Our scab-virus, when inoculated into calves dermally or intradermally, produced a papular eruption already in the 1st generation of calf-skin passage.(7) While the original scab-virus causes only a stimulating reaction on the inoculated skin of rabbits, the above-mentioned 1st rabbit-passage virus produced a characteristic red eruption, as large as the half size of red bean, on the skin of rabbits (Fig. 1). Similar eruption was still observed up to the 4th generation of rabbit-passage. The 5th transfer-virus, however, could first yield a papule-like eruption on the skin of rabbits (Fig. 2). The 4th transfer-virus did not yet cause any reaction on the cornea of rabbit, but it produced a papule-like eruption on the skin of calf. The 5th transfer-virus caused a clouding on the cornea of rabbits in 66%, but not yet on the cornea of calves, while it developed scattered pustules on the skin of calves.The rabbit, which has been inoculated with the 5th transfervirus, resisted the subsequent infection with vaccine virus. Then it may be considered that the 5th rabbit-transfer-virus began to lapinize, i. e., vaccinize. The 6th transfer-virus became decidedly virulent for rabbit and calf (Fig. 5), and it could develope confluent pustules on their skin. Moreover, it can produce a distinct clouding on rabbit's cornea, where we can easily detect the Guarnieri-bodies (Fig. 3). And it can also cause a clouding reaction. on the scarified cornea of calf.(8) The original scab-virus does not affect guinea-pigs at all.(9) The original scab-virus, like vaccine virus, could produce red eruptions on the skin of pigs.(10) The vaccination with the sensitized virus protected sheep, against reinoculation with this virus, as well as, with the sheeppox virus.(11) From the above-stated facts, it became known that this. scab-virus sent to us originated from a sheep-pox animal.

Decellularized Porcine Cornea-Based Hydrogels for Regeneration of Corneal Epithelium and Stroma

Active cell migration in retransplanted rat cardiac allografts during the course of chronic rejection

“Keep on ROCKIn”: Repurposed ROCK inhibitors to boost corneal endothelial regeneration

Cell migration is achieved by the dynamic feedback interactions between traction forces generated by the cell and exerted onto the underlying extracellular matrix (ECM), and intracellular mechano-chemical signaling pathways, e.g., Rho GTPase (RhoA, Rac1, and Cdc42) activities [1,2,3]. These components are differentially distributed within a cell, and thus the coordination between tractions and mechanotransduction (i.e, RhoA and Rac1 activities) must be implemented at a precise spatial and temporal order to achieve optimized, directed cell migration [4,5]. Recent studies have shown that focal adhesions at the leading edge exert strong tractions [6], and these traction sites are co-localized with focal adhesion sites [7]. Further, by using the fluorescence resonance energy transfer (FRET) technology coupled with genetically encoded biosensors, researchers reported that Rho GTPases, such as RhoA [8], Rac1 [9], and Cdc42 [10] are maximally activated at the leading edge, suggesting the leading edge of the cell as its common functional site for Rho GTPase activities. All these works, however, were done separately, and the relationship between tractions and mechanotransduction during cell migration has not been demonstrated directly because of the difficulty in simultaneously recording tractions and mechanotransduction in migrating cells, precluding direct comparison between these results. Furthermore, these studies have been conducted by monitoring cells on glass coverslips, the stiffness of which is ∼ 65 giga pascal (GPa), at least three to six order higher than the physiological range of ECM stiffness. Although it is increasingly accepted that ECM stiffness influences cell migration, it is not known exactly how physiologically relevant ECM stiffness (order of kPa range) affects the dynamics of RhoA and Rac1 activities. For a complete understanding of the mechanism of mechano-chemical signaling in the context of cell migration, the dynamics and interplay between biomechanical (e.g., tractions) and biochemical (e.g., Rho GTPase) activities should be visualized within the physiologically relevant range of ECM stiffness.

Temporal and spatial regulation of actin-based cytoskeletal organization and focal adhesion formation play an essential role in cell migration. Here, we show that tyrosine phosphorylation of a focal adhesion protein, paxillin, crucially participates in these regulations. We found that tyrosine phosphorylation of paxillin was a prominent event upon integrin activation during epithelial-mesenchymal trans-differentiation and cell migration. Four major tyrosine phosphorylation sites were identified, and two of them were highly inducible upon integrin activation. Paxillin exhibits three distinct subcellular localizations as follows: localization along the cell periphery colocalized with circumferential actin meshworks, macroaggregation at focal adhesions connected to actin stress fibers, and diffuse cytoplasmic distribution. Tyrosine phosphorylation of paxillin localized at the cell periphery and focal adhesions was shown using phosphorylation site-specific antibodies. Mutations in the phosphorylation sites affected the peripheral localization of paxillin and paxillin-containing focal adhesion formation during cell migration and cell-cell collision, accompanied by altered actin organizations. Our analysis indicates that phosphorylation of multiple tyrosines in paxillin α is necessary for the proper function of paxillin and is involved in the temporospatial regulation of focal adhesion formation and actin cytoskeletal organization in motile cells.