In vivo comparison of three different porous materials intended for use in a keratoprosthesis

Abstract

Aim: The goal was to compare the biological response of the corneal stroma with three porous materials: a melt blown microfibre web of polybutylene:polypropylene (80:20); a polyester spun laced fabric (polyethylene terephthalate), and an expanded polytetrafluoroethylene. Fifty per cent of each of the materials were modified using argon radio frequency.

Methods: Discs (6 mm in diameter) were inserted into interlamellar stromal pockets and followed for a period of 12 weeks. Clinical evaluations were performed weekly. At 6 and 12 weeks, fibroplasia and the distribution of matrix proteins and growth factors (bFGF and TGF-beta) were evaluated immunohistochemically. The characterisation of glycosaminoglycans was determined after selective extraction and digestion.

Results: The response to the disc resembled that of a wound with a decrease in keratan sulphate and an increase in dermatan sulphate. Pretreatment of the disc reduced corneal oedema and neovascularisation. Heparan sulphate, not normally detected in the corneal stroma, was detected in the region immediately surrounding the disc and in the discs of some materials. The presence of glycosaminoglycans and collagens in the disc indicated that cells had migrated into the disc and deposited a complex matrix in all three materials. The collagen response was not surface specific. bFGF and TGF-beta were detected in the region between the disc and the stroma in the polybutylene material and became diffuse with time.

Conclusion: Fibroplasia occurred most rapidly into the polyester discs but was accompanied by a large number of inflammatory cells. While the distribution of collagens was not altered by the material, the expression and distribution of growth factors was material dependent. bFGF was expressed transiently and occurred before that of TGF-beta. It is predicted that the transient expression of growth factors mediates the regulation of matrix proteins.

Figure 1

Oedema. (A) Non-treated. Corneal oedema gradually decreased over 6 weeks after surgery. Oedema was minimal after 12 weeks. Individual analysis at 6 weeks —polybutylene: 3/9 animals had a score of 1; polyester: 2/9 animals had a score of 2; and ePTFE: 3/9 animals had a score of 1. (B) Treated. Corneal oedema was minimal 8 weeks after treated discs were inserted. These discs were surface modified with an argon rf plasma. The oedema in response to the polyester disc decreased markedly after surface treatment. Individual analysis at 6 weeks—polybutylene: 1/9 animals had a score of 2 at 6 weeks; polyester: no animals had oedema; and ePTFE: 1/9 animals had a score of 2. Each data point represents the average of nine corneas.

Figure 2

Neovascularisation. (A) Non-treated. When discs were implanted, neovascularisation was detected within 1 week after surgery. After 3 weeks, blood vessels approached the disc margin. Neovascularisation was most extensive when polyester and polybutylene discs were inserted. In response to ePTFE, vessels were never detected in the discs and after 8 weeks some vessels had occluded. Individual analysis at 12 weeks —polybutylene: 5/9 animals had a score of 4; polyester: 9/9 animals had a score of 4; and ePTFE: 4/9 animals had a score of 4. (B) Treated. When ePTFE discs were surface treated neovascularisation increased while the response to the other two materials decreased. Neovascularisation was more gradual in response to the modified polybutylene, not reaching the margin of the disc until 4 weeks. Individual analysis at 12 weeks—polybutylene: 4/9 animals had a score of 4; polyester: 5/9 animals had a score of 4; and ePTFE: 9/9 animals had a score of 4. Each data point represents the average of nine corneas.

Figure 3

Histological appearance of discs inserted into stromal interlamellar pockets 12 weeks after surgery. Cross sections were stained with haematoxylin and eosin. (A) Polybutylene disc inserted (100×). (a) Higher magnification of (A) indicated by an asterisk (200×). (B) Polyester disc inserted (100×). (b) Higher magnification of (B) indicated by an asterisk (200×). A large number of inflammatory cells are present within the polyester disc. (C) ePTFE disc inserted (100×). (c) Higher magnification of (C) indicated by an asterisk (200×). In all cases the cells migrated from the anterior and posterior stroma into the disc. The data represent those from nine animals.

Figure 4

Distribution and composition of glycosaminoglycans. Keratan and dermatan sulphate were localised on cross sections of corneas 42 days after treated discs were inserted. (A), (D) Polybutylene disc inserted; (B), (E) polyester disc inserted; and (C), (F) ePTFE disc inserted. (A), (B), (C) Corneas stained with anti-dermatan sulphate shows dermatan sulphate within the disc. (D), (E), (F) Corneas stained with anti-keratan sulphate shows keratan sulphate within the disc (100×). Composition of glycosaminoglycans present 12 weeks after implantation of the porous materials. Aliquots from purified extracts were assayed for dermatan sulphate (DS), keratan sulphate (KS), and heparan sulphate (HS). Dermatan sulphate was defined as GAG susceptible to chondroitinase ABC. Keratan sulphate was defined as GAG susceptible to heparanase III. Data are presented as specific GAG/total GAG.

Figure 5

(A) Glycosaminoglycans present in tissue adjacent to the disc. (B) Glycosaminoglycans present in the disc. Heparan sulphate is detected after insertion of the disc in both the surrounding tissue and in the disc. Keratan sulphate is reduced in response to injury. Each data point represents data from nine pooled samples.

Figure 6

Distribution of bFGF and TGF-β 6 weeks after untreated discs were implanted in the cornea. (A), (D) Polyester discs inserted; (B), (E) polybutylene discs inserted; (C), (F) ePTFE discs inserted. (A), (B), (C) Corneas stained with a MAb to bFGF. bFGF is localised along the interface between the disc and stroma in (B) and (C) (arrow). (D), (E), (F). Corneas stained with a MAb to TGF-β. TGF-β is present along the margin of the disc and stroma in (E) (arrow) and is diffuse in (D) and (F) (100×).

Figure 7

Distribution of bFGF and TGF-β 12 weeks after untreated discs were inserted into the stroma. (A), (D) Polyester disc inserted; (B), (E) polybutylene disc inserted; and (C), (F) ePTFE disc inserted. (A), (B), (C) Localisation of bFGF. bFGF was present along the interface between the disc and stroma of (C) and was diffuse in (A) and (B). (D), (E), (F) Localisation of TGF-β. TGF-β was diffuse throughout the disc (100×).

Figure 8

Localisation of collagen types I, III, and VI in the cornea after polybutylene discs were inserted. Immunohistochemistry of corneal cross sections after 6 weeks (A, B, C) and 12 weeks (D, E, F). (A), (D) Localisation of type I collagen; (B), (E) type III collagen; and (C), (F) type VI collagen (100×).