Collagen Synthesized in Fluorocarbon Polymer Implant in the Rabbit Cornea

Exp. Eye Res. (1996) 62, 367–376

Collagen Synthesized in Fluorocarbon Polymer Implant in the Rabbit Cornea I S A B E L L E D R U B A I Xa, b*, J E A N-M A R C L E G E A ISa, c*, N A Y L A M A L E K-C H E H I R Ea, c, M I C H EA L E S A V O L D E L L Ia, M A U R I C E M E; N A S C H Ea, L A D I S L A S R O B E R Tb, G I L L E S R E N A R Da, c    Y V E S P O U L I Q U ENa, c INSERM U86, Laboratoire de Biochimie de la CorneU e, Paris, France, b Equipe de Biochimie des Tissus Conjonctifs, UniversiteU Paris VII, Paris, France and c DeU partement d’Ophtalmologie HoW tel-Dieu de Paris, France a

(Received Lund 12 June 1995 and accepted in revised form 17 November 1995) The integration of microporous polymer into tissues is of great interest for the production of keratoprosthetic devices. Our previous studies showed functional differentiated cells and collagen synthesis in the pore of an expanded polytetrafluoroethylene implant. This study identifies and quantifies collagen types synthesized in the implant. Expanded polytetrafluoroethylene polymers were implanted in the rabbit corneas. The collagen extracted from the polymer and implanted stroma after 1, 3 and 6 months was quantified by measuring hydroxyproline. The relative proportions of collagen types were determined by densitometric analysis after SDS–PAGE. The collagen-to-protein ratio in the polymer increased from 0±22 to 0±70 between the first and third month after implantation becoming similar to control cornea. So that of the protein and collagen densities in the polymer and implanted stroma were similar to the control from the third month. The collagen synthesized in the polymer was mainly type I (87 %) plus a small amount of type III (8 %) 1 month after implantation. The collagen distribution from the third month after implantation was similar to that of the controls and remained constant thereafter in the polymer implant and in the implanted stroma. Immunogold labelling techniques confirmed these results. Implantation of this PTFE disc induced no obvious modification of the corneal stroma, confirming that this polymer is a good interface that is compatible with the native corneal stroma. The keratocytes in this polymer rapidly adopted a corneal phenotype, distinct from the dermal or scaring phenotype as shown by the collagen types produced in the implant. # 1996 Academic Press Limited Key words : cornea ; biomaterials ; collagen ; expanded polytetrafluoroethylene ; keratoprosthesis ; immunogold labelling ; artificial cornea.

1. Introduction The integration of microporous polymers into tissues is of great interest for the production of keratoprosthetic devices. The peripheral prosthetic material should be incorporated into the biological substrate of the host. It should also have a porous structure able to accommodate stromal fibroblasts with maintained corneal phenotype. Various microporous polymers have been investigated. Chirila et al. (1993, 1994a, 1994b) used a porous phema, poly (2-hydroxyethylmethacrylate) as a skirt for an artificial cornea. Proplast, a porous polytetrafluoroethylene (PTFE), and vitreous carbon were used by Barber, Feaster and Priour (1980) as an implant in the rabbit cornea. Trinkaus-Randall et al. (1988, 1991, 1994) and Leibowitz et al. (1994) chose a porous structure, prepared from melt blown-polyolefins. The materials were invaded by stromal keratocytes both in vitro and in vivo. One of the most suitable materials for the skirt was a porous PTFE. Legeais et al. (1992) carried out a comparative study on PTFEs and showed that a thickness of 200 µm with parallel 50 µm pores * For correspondence at : Ho# tel-Dieu de Paris, INSERM U 86, 1 Place du Parvis No# tre-Dame, 75004 Paris, France.

0014–4835}96}040367­10 $18.00}0

specially devised by Impra was the most suitable biocompatible microporous PTFE, and that it became surprisingly translucent and wettable after its implantation in the stroma. This non-commercial PTFE seems to offer the best interface, with no evidence of encapsulation and without any corneal inflammation or vascularization. A recent study confirmed this result (Legeais et al., 1994) and revealed that the number of fibroblasts in the polymer increased with time, reached a maximal density at 3 months, and remained constant thereafter. Electron microscopic observations demonstrated that the collagen fibril organization was regular (Legeais et al., 1992, 1994). Thus, it was of special interest to determine the types of collagen synthesized during the organization of the new extracellular matrix within the pores of the implant. Our second objective was a biochemical evaluation of the healing characteristics of this ePTFE polymer implanted in the rabbit stroma as a corneal substitute. The collagen content was quantified by measuring hydroxyproline. The collagen types were identified by SDS–PAGE and by immunogold labelling. The three major collagen types in the corneal stroma, I, V and VI (Davison, Hong and Cannon, 1979 ; Newsome, Gros and Hassell, 1982 ; Zimmermann et al., 1986 ; Kern, # 1996 Academic Press Limited

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Menasche and Robert, 1991) were thus investigated. The study focused on type III collagen, as this is found in developing (Conrad, Dessau and Von Der Mark, 1980 ; Tseng, Smuckler and Stern, 1982 ; Lee and Davison, 1984 ; Cintron et al., 1988) and healing tissues (Cintron et al., 1978 ; Cintron, Hong and Kublin, 1981 ; Hanna et al., 1989 ; Malley et al., 1990 ; Sundar Raj et al., 1990). 2. Materials and Methods Experimental Animals This study complied with the ARVO resolution on the use of animals in research. A total of 18 adult New Zealand albino rabbits weighing 2±5–3 kg were anaesthetized with intramuscular injections of Narcozep (0±05 ml kg−") and Ketamine (1±5 mg kg−"). Expanded polytetrafluoroethylene implants were specifically devised by Impra Ltd. (Arizona) (0±2 mm thick, 50 µm pore size with a mean porosity of 88³4 %, and the channels were perpendicular to the surface). All the polymer implants used in this study were from the same lot (E 208) and were cleaned and sterilized by the same procedure. The 6 mm diameter discs were implanted after a free-hand intralamellar dissection (Legeais et al., 1992, 1994). The cornea was closed with 10±0 nylon sutures and the knots buried. No steroids were applied topically. The animals were followed periodically by slit lamp biomicroscopy for up to 6 months. The animals were killed with a lethal intracardiac injection of sodium pentobarbital 1, 3 or 6 months after implantation. Histology The ocular surface was flushed with 2±5 % glutaraldehyde in 0±1  Cacodylate buffer. The corneas, together with a 2 mm rim of sclera, were removed, immersed in the fixative for 2 hr, and prepared for light and transmission electron microscopy. The tissue was stored overnight in buffer at 4°C, exposed to 2 % osmium tetraoxide for 2 hr, dehydrated in alcohol, and embedded in a plastic medium. Plastic sections (1 µm thick) were stained with Toluidine Blue. Paraffin sections were stained with Sirius red and examined under a light microscope (Legeais et al., 1994). Thin sections of corneas with implanted polymer were stained with uranyl acetate–lead citrate and examined in a Philips CM 10 transmission electron microscope. Biochemistry Corneas were removed at 1 month, 3 months and at 6 months (as a control of maximal colonization) (Legeais et al., 1994). Corneas were carefully dissected free of epithelium and endothelium and the polymer implants were removed. Polymer implant, implanted stroma and two control stromas were solubilized with

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pepsin [Sigma, 3000 U (mg solid)−"] (3 mg per stroma and 1 mg per polymer implant) in 0±5  acetic acid at room temperature for 24 hr. This procedure totally solubilized the stroma giving a crude extract. We ascertained that the polymer implant was free from collagen by TEM and hydroxyproline analysis. The hydroxyproline content of the crude pepsin-solubilized extracts was determined as described by Bergman and Loxley (1963) and converted to collagen content using the factor of 8±33 calculated on the basis of an average hydroxyproline content of 12 % in mammalian collagens. Total protein was determined by the Lowry procedure (1951). The optical density of noncollagen proteins was differentiated from that of collagen by using bovine serum albumin and collagen as standards plus appropriate amounts of pepsin. Total protein and collagen densities were evaluated using a mean volume of 34 mm$ for implanted and control stroma (12 mm diameter, 300 µm thick) and of 5 mm$ for the polymer implant (6 mm diameter, 200 µm thick and a mean porosity of 88 %). Collagen types were separated by SDS polyacrylamide gel electrophoresis (4±4 % stacking gel and 7±5 % separating gel) in a Miniprotean II (Biorad) by the method of Laemmli (1970). Samples were reduced with 5 % beta-mercaptoethanol and heated at 95°C for 10 min prior to loading. Types I, III and V collagens from human placenta (Sigma) were used as standards. The gels were silver stained (Silver stain kit, Biorad), and analysed using an automated image analyser (Biocom) with specialized software (Lecphor). Collagen types were quantified using the relative band densities of the α chain polypeptides as previously described (Kern et al., 1991). Briefly, the type I was quantified by the sum of α1 and α2 chains (arrows C and D on the gel, apparent molecular weight : 116 and 99 kDa, respectively). To quantify type V collagen we added α1 plus α2 chains (arrows A and B on the gel, apparent molecular weight : 140 and 125 kDa, respectively). For type VI collagen we summed α1 plus α2 plus α3 chains (arrows E, F and G on the gel, apparent molecular weight : 63, 56 and 49 kDa, respectively). Immunogold Labelling Corneas were prepared for immunogold labelling after a follow-up of 3 months (Malek et al., 1993). The samples were fixed with 4 % paraformaldehyde in freshly prepared 0±1  pH 7±3 cacodylate buffer for 22–24 hr at ­4°C. Blocks containing the implanted ePTFE were cut and soaked in 0±15  glycine for 1 hr. They were then processed for embedding in L. R. White (London Resin Company, U.K.) at ®20°C. Curing of the resin was done with an accelerator at the same temperature. Ultrathin sections were done and mounted on Parlodion-carbon-coated Nickel 200 mesh grids. Sections were preincubated in urea. The buffer used for dilution of labelling reagents was PBS–BSA–gelatin–Tween. Incubation with the pri-

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mary antibody was done for 2 hr in a well-humidified atmosphere at room temperature. Primary antibodies for collagen type I were goat anti-human-Ig G from Institut Pasteur (Lyon, France) and diluted 1}15 or 1}30. For collagen type III labelling, we used goat anti-human-Ig G (1}50) from Southern Biotechnology Associates. Sections were rinsed then incubated with the secondary antibody for 45 min. We used 10-nm colloidal gold conjugated rabbit anti-goat-IgG (BioCell). Sections were rinsed several times and then post-fixed with 2 % glutaraldehyde for 2 min. Staining for TEM observation was done after uranyl acetate and lead citrate staining. Statistics Biochemical assays were carried out at least in triplicate. All values are reported as means³... Statistical analysis was performed using Student’s unpaired t-test. We compared the polymer implant and the implanted stroma with control stroma. 3. Results Clinical, Histological and Ultrastructural Observations The white opaque implanted polymer became translucent in the first 2 weeks, as previously described [Fig. 1(A)]. No corneal deposits or vessels were observed. The epithelium remained normal without thinning of the central epithelium in the front of the microporous polymer. The endothelium appeared to be normal in all specimens. There was no evidence of corneal stromal inflammation or corneal melting around the polymer implant after the first month. No keratocytes were seen along the tissue–plastic interface and the anterior and posterior surfaces of the implants appeared free of lipid deposits. The polymer implants appeared to be filled with keratocytes in a vertical position [Fig. 1(B) and (C)]. Sirius red staining of paraffin sections revealed dense staining for collagen [Fig. 1(D)]. Electron microscopie examination showed no vacuolization of cells, but they had dilated endoplasmic reticulum and mitochondria. The collagen formation fibrils were organized in a regular pattern [Fig. 2(A)]. These results are consistent with those previously described (Legeais et al., 1992, 1994). Protein and Collagen Densities The small amount of tissue available from the polymer implants made it necessary to quantify and identify collagen in crude pepsin-solubilized extracts. The polymer implant, and implanted and control stromas were processed identically and simultaneously. The pepsin solubilization experiments were first carried at 4°C (Cintron et al., 1981 ; Kern et al., 1991). This solubilized only 75 % of the total collagen. The collagen types are known to differ in their susceptibility to pepsin solubilization (Cintron et al.,

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1981 ; Kern et al., 1991). By contrast, incubation with pepsin at room temperature for 24 hr resulted in total solubilization of corneal stroma, with no difference between implanted and control corneal stromas. This procedure removed all the collagen in the pores of the PTFE implants. Electron microscopy [Fig. 2(B)] showed that the pores of the PTFE disc appeared free of native collagen fibrils. Hydroxyproline assay indicated that over 95 % of total collagen was removed from the PTFE implants. Total protein and collagen are expressed as densities ( µg mm−$) to quantitatively compare polymer implant and implanted stroma to control (Fig. 3 and Fig. 4). Volumes were estimated as described in Materials and Methods. The total protein density in the polymer was half that of the control (P ! 0±001) 1 month after implantation and the collagen density was five-fold lower (P ! 0±001). Total protein and collagen densities in the polymer implant and in implanted stroma were very close to those of controls from the third to the sixth month. Some of our recent studies have shown that the collagen density depends upon the permeability of the pore of the polymer (Briat et al., 1995). The permeability and porosity of the polymer used in this study gave the highest protein and collagen contents within the biomaterial. The collagen-to-total protein ratios in the Impra disc, implanted stroma and control stroma are shown in Fig. 5. The collagen-to-total protein ratio in the Impra disc increased from 22 % 1 month after implantation to 70 % after 3 and 6 months. Thus, 1 month after the implantation the polymer implant was mainly filled with non-collagen proteins, whereas from the third month collagen and non-collagen proteins were present in the same proportion as in the extracellular matrix of control stroma. The collagen-to-protein ratio (70 %) in implanted stroma, remained similar to that in control stroma from the first to the sixth month after implantation. However, because the analyses were carried out on the whole implanted stroma, small changes may have occurred at the interface between the polymer and the stroma. SDS–PAGE and Densitometric Analysis To identify collagen types after SDS–PAGE, preliminary experiments were carried out to determine the optimal conditions. With a prior collagenase treatment, no bands were detected, except for a dark mark at the bottom of the gel consisting of pepsin. This assured us that bands in our gels contain only collagen peptide. To detect type III collagen, a minor component of cornea, we used the sensitive silver stain procedure. Serial dilutions prior to the loading indicated us that 2 µg of total collagen calculated from hydroxyproline was the minimal quantity allowing the detection of type III collagen without much overstaining of type I collagen, the major component of the cornea. The collagen types in crude pepsin-

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F. 1. Rabbit corneal stroma implanted with a 6 mm diameter Impra disc. Clinical appearance, Day 7 (A). Cell distribution and cell densities in the polymer implant, 3 months [(B) original magnification ¬320]. [(C) original magnification ¬800] ; Sirius red staining, 3 months [(D) original magnification ¬800]. K, keratocyte ; P, pore ; S, stroma.

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F. 2. TEM. Collagen synthesis by fibroblasts penetrating into polymer implant pores : before pepsin treatment [(A) original magnification ¬31 000] and after pepsin treatment [(B) original magnification ¬3300]. Arrow indicated collagen fibril. K, keratocyte ; P, pore.

Protein density (µg mm –3)

400 Control

Impra

Implanted stroma

300

200

*

100

0

1 3 6 1 Post-implantation time (months)

3

6

F. 3. Total protein density in the Impra disc and implanted stroma after 1, 3 and 6 months of implantation and in the control corneal stroma. * P ! 0±001 as compared to control corneal stroma.

solubilized extracts were identified after SDS–PAGE (Fig. 6). Adult control corneal stroma (lane 4) had the typical pattern of collagen type I with appreciable

amounts of collagen type V and VI. Collagen type III appeared mainly as a thin band between the α1 and the α2 bands of collagen type V. There may have been

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Collagen density (µg mm –3)

300 Control

Impra

Implanted stroma

200

100

* 0

1 3 6 1 Post-implantation time (months)

3

6

F. 4. Collagen density in the Impra disc and implanted stroma after 1, 3 and 6 months of implantation and the control corneal stroma. Results are expressed as µg mm−$. Collagen content was calculated from the hydroxyproline content as described in Materials and Methods. * P ! 0±001 as compared to control corneal stroma.

Collagen to protein ratio (%)

100 Control

Impra

Implanted stroma

80 60 40

* 20 0

1 3 6 1 Post-implantation time (months)

3

6

F. 5. Collagen-to-total protein ratios in pepsin-solubilized fractions from Impra discs and implanted stroma after 1, 3 and 6 months of implantation and control corneal stroma. * P ! 0±001 as compared to control corneal stroma.

A B C D

E F G

1

2

3

4

5

6

7

8

9

10

F. 6. SDS–PAGE of pepsin-solubilized collagens from control corneal stroma (lane 4), Impra discs after 1, 3 and 6 months of implantation (lanes 5, 6 and 7) and implanted stroma after 1, 3 and 6 months of implantation (lanes 8, 9 and 10). Samples were reduced with 5 % β mercaptoethanol. Wells were loaded with about 2 µg collagen, and proteins were silver stained. Molecular weight standards (Biorad) were 205, 116, 97, 66 and 45 kDa (arrows on the left, top to bottom). Collagen standards : Type I, III and V (lanes 1, 2 and 3). The major collagen α bands are indicated by arrows on the right : A : α1(V), B : α2(V), C : α1(I), D : α2(I), E : α1(VI), F : α2(VI), G : α3(VI). The experiment shown is representative of five separate experiments.

a second band comigrating with the α1 band of type I collagen. The dark mark at the bottom of the gel is pepsin (apparent molecular weight : 39 kDa). Although the same total collagen quantity (2 µg) was loaded on the gel, the lane 5 containing pepsin

extract from the polymer one month after the implantation appeared underloaded. We could not exclude a partial degradation of collagen resulting in bands migrating at the bottom of the gel. One month after the implantation the polymer implant (lane 5)

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T I Relative amounts ( percentage of total) of type I, III, V and VI collagens in pepsin-solubilized extracts isolated from polymer implants, implanted stroma and control corneal stroma at various times after implantation Polymer implant

Type I

Implanted stroma

Control

1 month

3 months

6 months

1 month

3 months

6 months

58³5

87³7 *** 8³6 *** 3³3 *** 1³1 ***

52³10 ns 5³2 ns 19³9 ns 24³6 ns

55³10 ns 2³2 ns 19³7 ns 24³9 ns

50³10 ns 2³2 ns 20³5 ns 28³8 ns

57³7 ns 4³3 ns 17³7 ns 22³7 ns

58³7 ns 2³2 ns 15³5 ns 25³10 ns

Type III

3³2

Type V

15³6

Type VI

24³4

Densitometric analyses were performed as described in Materials and Methods. *** P ! 0±001, ns, not significant (P " 0±05) as compared to control corneal stroma.

was filled mainly with type I collagen. There were only small amounts of type III collagen, detected as a band migrating between the α1 and the α2 bands of collagen type I. This different type III collagen location from those in control was also reported by Conrad et al. (1980). Type V and VI collagens were almost undetectable. The collagen pattern in the polymer implant three and six months after the implantation (lanes 6 and 7) as well as in implanted stroma (lanes 8, 9 and 10 respectively), was similar to that of the control stroma. Despite the fact that densitometric analysis after SDS–PAGE are not quantitatively accurate, this technique gives real indications of the distribution of collagens. Densitometric analysis was used to determine the relative proportion of each collagen type (Table I). In agreement with others (Cintron et al., 1981 ; Newsome et al., 1982 ; Zimmermann et al., 1986 ; Kern et al., 1991), types I, V and VI were the three major collagens in adult corneal stroma, accounting for 58³5 %, 15³6 % and 24³4 % of the total collagen. Although it could have been underestimated, type III collagen was a minor component of the adult corneal stroma (3³2 %). Unlike the other collagen types, type III collagen was not stained by Coomassie blue. Only two collagen types were detected in the polymer implant 1 month after implantation, type I collagen was the main component (87³7 %), with significant amounts of type III collagen (8³6 %), and very little type V and type VI collagens (less than 3 %). The polymer contained slightly less type I collagen (52³10 vs. 58³5) and increased percentage of type III collagen (5³2 vs. 3³2) than the control 3 months after implantation. But these differences were not statistically significant (P ¯ 0±068 for type I, and P ¯ 0±052 for type III). There was no further change in the relative percentage of type V and VI collagens (P " 0±05). Six months after the implantation, the relative amounts of type I, V, VI and III collagens within the pores of the Impra disc were similar to those

in adult control stroma six months after implantation (P " 0±05). The collagen type distribution in implanted corneal stroma remained close to that of controls at all later times. There was a non-significant (14 %) decrease in collagen type I (P ¯ 0±062), without any increase in type III collagen only 1 month after implantation. Immunogold Labelling After the specific type I collagen immunogold labelling, we observed inside the polymer an intense staining only on the collagen fibrils and none between fibrils [Fig. 7(A)]. After 3 months, there was a scarce labelling of the collagen fibrils with anticollagen III only inside the polymer and negative elsewhere [Fig. 7(B)]. As expected, labelling was present in the surrounding stroma with anticollagen I and no gold beads were found on the resin and endothelium [Fig. 7(C)] and on epithelium with both anticollagen [Fig. 7(D)]. 4. Discussion Previous studies have demonstrated that highporosity ePTFE implants without external wrapping provide a satisfactory interface with the corneal stroma. This opaque crystalline polymer becomes translucent and wettable after its implantation and collagen fibrils formed within its pores. This study shows that the maximal total protein and collagen densities in PTFE implant were similar to those in control stroma by the third month, consistent with the maximal keratocyte density (Legeais et al., 1994). SDS–PAGE and densitometric analysis revealed similar patterns of collagen type distribution in the polymer implant and the control stroma. Immunogold labelling techniques confirmed these results and clearly identified type I collagen to be the predominant synthesized collagen in the polymer. Type III collagen was barely noticeable after 3 months confirming the results

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F. 7. Immunogold labelling. (A) Labelling of the collagen fibrils in the polymer with anticollagen I ; (" 3 months) ; dilution : (1}30), (original magnification ¬61 560) ; (B) anticollagen III labelling in the polymer (1}50), (original magnification ¬61 560). Almost no dots were observed on the collagen fibrils [(C) and (D)], no labelling is observed with anticollagen I and III in resin, endothelium and epithelium. (original magnification ¬54 400). EP, epithelium ; R, resin ; ST, stroma ; END, endothelium ; arrow, gold beads.

obtained biochemically (Malek et al., 1993). However, further studies should be devised for all major collagen types using other techniques such as Western blot analysis or immunoprecipitations. The three major collagen types were type I, with appreciable amounts of type V and VI collagens.

Despite numerous biochemical and immunohistochemical investigations, the presence and location of type III collagen in corneal tissue has not yet been clearly elucidated. Type III collagen seems to be mainly synthesized during the development of the corneal stroma in the rabbit and in the formation of

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scar tissue in the adult rabbit (Schmut, 1977 ; Cintron et al., 1978, 1988). Although type III collagen was found to be a minor component of adult rabbit corneal stroma (less than 3 % of total collagen) it was detectable by silver staining. This is consistent with the reported transient increase in type III collagen in growing tissue. As suggested by Cintron et al. (1988), the intermolecular disulphide cross-links of type III collagen would provide mechanical stability during tissue growth. The fibroblastic cells penetrating into the pores were viable and produced a new collagen matrix with a composition and structure very similar to that of control corneal stroma. This shows the capacity of keratocytes, even after their migration into the polymer implant, to establish and maintain the typical corneal phenotype producing original highly organized collagen matrix. This agrees with Doane et al. (1992) who showed that corneal fibroblasts grown within a three-dimensional collagen gel could assemble and deposit small diameter fibrils with a collagen composition and structure identical to that of the secondary stroma. Corneal transparency is thought to depend on the maintenance of uniform diameter and orientation of collagen fibrils as well as regular interfibrillar spaces (Maurice, 1969 ; Benedek, 1971). The collagen fibril diameter in mature corneal stroma depends on the type I}type V ratio (Birk et al., 1990), whereas type VI collagen and non-collagen proteins and proteoglycans located between fibrils maintain a regular interfibrillar spacing (Hassell et al., 1983 ; Bonaldo et al., 1990). We showed that this chemically well-defined crystalline opaque fluorocarbon became transparent after implantation (Legeais et al., 1992, 1994), and had a refractive index (1±376) identical to the normal adult cornea (Spezati, 1989). The transparency of the PFTE implant in the first month after implantation could be due to the predominance of the non-collagen proteins whose refractive index is 1±354 (Maurice, 1969), close to 1±376. The collagen matrix subsequently formed in the microporous meshwork is thus very similar to that of the control corneas and explains the permanent transparency of the implanted polymer. Clinical, histological and biochemical analyses, indicated no abnormalities of corneal healing due to the implantation of the polymer in the rabbit corneal stroma. To our knowledge, this is the first demonstration that fibroblastic cells migrating into a polymer implanted in corneal stroma synthesize a collagen matrix very similar to that of control. This polymer has now been used for 2 years in humans as an optical support for keratoprosthesis with high rates of success (Legeais et al., 1995).

Acknowledgements None of the authors have proprietary of financial interests in any of the devices described herein. Author JML is named

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as inventor on a patent which, following the French law is solely and fully owned by France Chirurgie Industries, S.A., Paris. This work was supported by the De! le! gation a' la Recherche Clinique, Fondation de l’Avenir, The French Institute for Medical Research (INSERM). Presented in part at the annual ARVO Meeting 1995 (Fort Lauderdale, FL, U.S.A.).

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