J.H. Fitton

Cell viability and inflammatory response in hydrogel sponges implanted in the rabbit cornea

In the quest for the development of a functional keratoprosthesis, the biocompatibility of the porous skirt material in the Chirila keratoprosthesis (KPro) was investigated. The population of live and dead cells within, and the inflammatory response to, a tissue-integrating poly(2-hydroxyethyl methacrylate) (PHEMA) sponge were studied. Samples of the hydrogel sponge were implanted in rabbit corneas and explanted at predetermined time points up to 12 weeks. The explanted sponges were subjected to cell viability assay using two types of fluoroprobes, 5-chloromethylfluorescein diacetate and ethidium homodimer-1. A semiquantitative analysis was performed to assess the number of dead cells within the sponge and in the area of corneal stroma proximal to the sponge. Five rabbits were used for each end point (2, 4 and 12 weeks). To investigate the inflammatory response to the sponge, immunocytochemistry, using specific antibodies to rabbit macrophages, enzyme histochemistry of chloroacetate esterase (to detect neutrophils) and transmission electron microscopy (TEM) were also employed at 24 h, 2, 4 and 12 weeks after implantation. Four weeks after implantation, fewer viable cells were observed in the sponge when compared to the 2-week implant. However, the proportion of viable cells increased dramatically by 12 weeks. The proportion of nonviable cells decreased gradually with time; central sponge contained 34+/-11 % dead cells after 2 weeks, and 15+/-4.3% after 12 weeks. The staining of inflammatory cells demonstrated the presence of macrophages and neutrophils up to 12 weeks after implantation. TEM confirmed the presence of these cell types and others. including eosinophils and myofibroblasts, as well as blood capillaries. The presence of a significant number of viable cells at each time point and the uniform reduction of the nonviable cell proportion with time suggests that the sponge is a conducive environment supporting a prolific, viable cellular colonization. Dead cells observed in the first instance indicate a normal injury pattern. However, the presence of a small but significant proportion of invading inflammatory cells 12 weeks after implantation confirms a characteristic pattern of wound healing within the sponges.

Assessment of anticollagenase treatments after insertion of a keratoprosthetic material in the rabbit cornea

Purpose This study was performed to evaluate the enzyme production in response to implantation of the hydrogel material used in the experimental Chirila keratoprosthesis (KPro) and to assess the effects of five topical drugs on enzyme production and activity. KPros may be extruded from the cornea as a result of tissue melting, a process that involves excessive enzyme activity. To reduce the possibility of implant loss for the hydrogel Chirila KPro, a number of antiinflammatory drugs that have been used to treat other corneal melting conditions were investigated for their effect on initial collagenase activity after the implantation of KPro material into the rabbit cornea. Methods Poly(2-hydroxyethyl methacrylate) sponge pieces were implanted into rabbit corneas. Prednisolone, tetracycline, medroxyprogesterone, acetylcysteine, and sodium citrate were assessed for effects on gelatinolytic activity and stromal collagenase [matrix metalloprotease-1 (MMP-1)] production in vivo and in vitro by using zymography and Western blotting techniques. Results Whereas all five anticollagenase drugs were effective in reducing gelatinolytic activity in vitro, many were ineffective in vivo. However, medroxyprogesterone caused a reduction of gelatinolytic activity in vivo. The amount of MMP-1, as measured by immunoblotting, also was reduced by medroxyprogesterone treatment when compared with untreated controls. An increase in the apparent molecular weight of MMP-1 in operated corneas appears to be the result of the association of MMP-1 with collagen fragments resulting from the surgical trauma. Conclusion This study indicates that topical medroxyprogesterone may be a useful adjunctive therapy after prosthokeratoplasty.

Histologic Evaluation During Healing of Hydrogel Core-and-skirt Keratoprostheses in the Rabbit Eye

Purpose We developed two models that are modifications of our original poly(2-hydroxyethyl methacrylate) (PHEMA) core-and-skirt keratoprosthesis. In these keratoprostheses. the mechanical strength of the skirt has been considerably increased with divinyl glycol (DVG) as a cross-linking agent during polymerization. In one (KPro I). methyl methacrylate (MM A) was added as comonomer to increase cell adhesion, and in the other (KPro II). HEMA was polymerized with DVG without comonomer. The aim of this study was to evaluate the process of healing and biocolonization and to ascertain whether KPro I demonstrates better ingrowth than the mechanically stronger KPro II, after implantation in rabbit eyes. Methods Ten rabbits were used for each model and studied at five predetermined end points up to 26 weeks. The device was implanted as a full-thickness keratoprosthesis covered with a conjunctival flap. Results Neither prosthesis demonstrated extrusion or retroprosthetic membrane formation. There was no significant difference between the two types of prosthesis with respect to tissue ingrowth and surrounding tissue melting. Histologically. inflammation was not severe, but calcification was seen in most specimens. Evidence of biodegradation of the prosthesis also was seen. Conclusion In our original keratoprosthesis, fibrovascular invasion had occurred into the prosthetic skirt, but wound dehiscence and low mechanical strength resulted in an unfavorable outcome. In this series, the mechanical properties were improved, and KPro II was stronger than KPro I. Therefore KPro II would be the preferred polymer combination for surgical manipulation. However, biodegradation and calcification require further investigation into the degree and significance of these adverse reactions.

The modulation of cellular responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases macrophage collagenase production in vitro

We examined the regulation of collagenase production by the monocyte/macrophage THP-1 cell line when these cells were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies. Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response. Copolymer hydrogels containing 2-ethoxyethyl methacrylate (EMA) or methyl methacrylate (MMA) also induced a high response, while PHEMA hydrogels induced a low level response and the phosphorylated hydrogel induced no response. This pattern was altered when the morphology of the hydrogels was changed to that of a sponge. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels. Sponges containing EMA and MMA produced low level response relative to the TCP control. PHEMA and phosphorylated sponges produced little and no response respectively. The dramatically reduced enzyme response to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge.