Non-mulberry Silk Fibroin Biomaterial for Corneal Regeneration

Successful repair of a damaged corneal surface is a great challenge and may require the use of a scaffold that supports cell growth and differentiation. Amniotic membrane is currently used for this purpose, in spite of its limitations. A thin transparent silk fibroin film from non-mulberry Antheraea mylitta (Am) has been developed which offers to be a promising alternative. The silk scaffolds provide sufficient rigidity for easy handling, the scaffolds support the sprouting, migration, attachment and growth of epithelial cells and keratocytes from rat corneal explants; the cells form a cell sheet, preserve their phenotypes, express cytokeratin3 and vimentin respectively. The films also support growth of limbal stem cell evidenced by expression of ABCG2. The cell growth on the silk film and the amniotic membrane is comparable. The implanted film within the rabbit cornea remains transparent, stable. The clinical examination as well as histology shows absence of any inflammatory response or neovascularization. The corneal surface integrity is maintained; tear formation, intraocular pressure and electroretinography of implanted eyes show no adverse changes. The silk fibroin film from nonmulberry silk worms may be a worthy candidate for use as a corneal scaffold

Pirfenidone nanoparticles improve corneal wound healing and prevent scarring following alkali burn.

To evaluate the effects of pirfenidone nanoparticles on corneal re-epithelialization and scarring, major clinical challenges after alkali burn.Effect of pirfenidone on collagen I and α-smooth muscle actin (α-SMA) synthesis by TGFβ induced primary corneal fibroblast cells was evaluated by immunoblotting and immunocytochemistry. Pirfenidone loaded poly (lactide-co-glycolide) (PLGA) nanoparticles were prepared, characterized and their cellular entry was examined in primary corneal fibroblast cells by fluorescence microscopy. Alkali burn was induced in one eye of Sprague Dawley rats followed by daily topical treatment with free pirfenidone, pirfenidone nanoparticles or vehicle. Corneal re-epithelialization was assessed daily by flourescein dye test; absence of stained area indicated complete re-epithelialization and the time for complete re-epithelialization was determined. Corneal haze was assessed daily for 7 days under slit lamp microscope and graded using a standard method. After 7 days, collagen I deposition in the superficial layer of cornea was examined by immunohistochemistry.Pirfenidone prevented (P<0.05) increase in TGF β induced collagen I and α-SMA synthesis by corneal fibroblasts in a dose dependent manner. Pirfenidone could be loaded successfully within PLGA nanoparticles, which entered the corneal fibroblasts within 5 minutes. Pirfenidone nanoparticles but not free pirfenidone significantly (P<0.05) reduced collagen I level, corneal haze and the time for corneal re-epithelialization following alkali burn.Pirfenidone decreases collagen synthesis and prevents myofibroblast formation. Pirfenidone nanoparticles improve corneal wound healing and prevent fibrosis. Pirfenidone nanoparticles are of potential value in treating corneal chemical burns and other corneal fibrotic diseases

Surface morphology of NPs: (A) AFM and (B) TEM pictures show smooth surface of the NPs.

<p>Surface morphology of NPs: (A) AFM and (B) TEM pictures show smooth surface of the NPs.</p

Collagen 1 expression was reduced in the pirfenidone NP treated cornea: Immunohistochemistry shows increased expression of collagen I in the superficial layer of corneas from control and free pirfenidone treated rats, and a reduced expression in the corneas from pirfenidone NP treated rats.

<p>Corresponding DAPI stained sections are also presented.</p

Pirfenidone prevents α-SMA expression in corneal fibroblasts: Western blot analysis shows reduction in α-SMA expression by pirfenidone from the TGF β induced corneal fibroblast in a dose dependent manner; Lane1- Untreated, Lane2- TGF β, Lane3- TGF β and 2.5 µg/ml free pirfenidone, Lane4- TGF β and 5 µg/ml free pirfenidone, Lane5- TGF β and 10 µg/ml free pirfenidone.

<p><i>Data is expressed as Mean ± SEM, N = 3, * p<0.05 vs Untreated, # p<0.05 vs TGF β.</i></p

Pirfenidone suppresses Collagen I expression in corneal fibroblasts: (A) Western blot analysis shows reduction of Collagen I expression by pirfenidone in TGF β induced corneal fibroblasts in a dose dependent manner; Lane1- Untreated, Lane2- TGF β, Lane3- TGF β and 2.5 µg/ml free pirfenidone, Lane 4- TGF β and 5 µg/ml free pirfenidone, Lane5- TGF β and 10 µg/ml free pirfenidone.

<p><i>Data is expressed as Mean ± SEM, N = 3, * p<0.05 vs Untreated, # p<0.05 vs TGF β.</i> (B) Western blot also shows that suppression of collagen I was similar in free pirfenidone and pirfenidone NP treated corneal fibroblasts; Lane1- Untreated, Lane2- TGF β, Lane3- TGF β and 5 µg/ml free pirfenidone, Lane4- TGF β and 5 µg/ml pirfenidone NP, Lane5- TGF β and 10 µg/ml free pirfenidone, Lane6- TGF β and 10 µg/ml pirfenidone NP. <i>Data is expressed as Mean ± SEM, N = 3, * p<0.05 vs Untreated, # p<0.05 vs TGF β.</i> (C) Imunocytochemistry also shows suppression of collagen I expression by pirfenidone from TGF β induced corneal fibroblasts in a dose dependent manner. The suppression was similar in free pirfenidone and pirfenidone NP treated cells.</p

Characteristics of nanoparticles.

<p><i>Data expressed as Mean ± SD</i>.</p

Effect of free pirfenidone and Pirfenidone NPs on corneal healing after alkali exposure: (A) Representative pictures of control, free pirfenidone and pirfenidone NP treated corneas after 1 hr, 4 th day and 7 th day of alkali treatment.

<p>(B) Corneal re-epithelialization time: Corneal re-epithelialization time was significantly (P<0.05) reduced in pirfenidone NP treated eyes compared to the control eyes, but the corneal re-epithelialization time in free pirfenidone treated eyes was not significantly different from the control eyes. <i>Data is expressed as Mean ± SEM, N = 6.</i> (C) Corneal haze: A significant (P<0.05) reduction in corneal haze score was evidenced in pirfenidone NP treated eyes compared to the control eyes. <i>Data is expressed as Mean ± SEM, N = 6.</i></p