RESUMEN
We examined the regulation of collagenase production by rabbit keratocyte, epithelial and mixed keratocyte/epithelial cell cultures which were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies (sponge and homogeneous gels). Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response with all cell culture types. Copolymer homogeneous gels containing 2-ethoxyethyl methacrylate (EEMA) or methyl methacrylate (MMA) induced a high response in keratocyte cultures, whilst PHEMA hydrogels induced a moderate response and the phosphorylated PHEMA (phos-PHEMA) hydrogel induced no response. Epithelial cells cultured on PHEMA, copolymer and phos-PHEMA hydrogels produced less collagenase activity than the keratocyte cells. The profile of collagenases produced by epithelial cells in response to phos-PHEMA was different to that for the other hydrogels. Co-cultured cells produced higher levels of collagenase (relative to the TCP) in response to hydrogels than did either the keratocytes or epithelial cells alone, but the response of phos-PHEMA was still the lowest. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels, although this effect was less prominent in the keratocyte cultures. The markedly reduced and alternative collagenase responses to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge and morphology.
Asunto(s)
Materiales Biocompatibles , Colagenasas/biosíntesis , Córnea/enzimología , Células Epiteliales/enzimología , Queratinocitos/enzimología , Polihidroxietil Metacrilato , Animales , Caseínas/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular , Técnicas de Cocultivo , Córnea/citología , Células Epiteliales/citología , Hidrogeles , Implantes Experimentales , Queratinocitos/citología , Inhibidores de la Metaloproteinasa de la Matriz , Fosforilación , Conejos , Propiedades de SuperficieRESUMEN
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.
Asunto(s)
Materiales Biocompatibles/química , Colagenasas/biosíntesis , Hidrogeles/química , Macrófagos/enzimología , Polihidroxietil Metacrilato/química , Línea Celular , Células Inmovilizadas , Inducción Enzimática , Humanos , Hidrogeles/farmacología , Macrófagos/efectos de los fármacos , Metacrilatos/química , Metilmetacrilato/química , Monocitos/efectos de los fármacos , Monocitos/enzimología , Fosforilación , Prótesis e Implantes , Propiedades de Superficie , Acetato de TetradecanoilforbolRESUMEN
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.
Asunto(s)
Córnea/efectos de los fármacos , Implantes Experimentales , Inhibidores de la Metaloproteinasa de la Matriz , Acetilcisteína/administración & dosificación , Acetilcisteína/farmacología , Administración Tópica , Animales , Western Blotting , Citratos/administración & dosificación , Citratos/farmacología , Colagenasas/metabolismo , Córnea/enzimología , Córnea/cirugía , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Gelatinasas/metabolismo , Supervivencia de Injerto , Medroxiprogesterona/administración & dosificación , Medroxiprogesterona/farmacología , Metacrilatos , Soluciones Oftálmicas , Prednisolona/administración & dosificación , Prednisolona/farmacología , Conejos , Citrato de Sodio , Tetraciclina/administración & dosificación , Tetraciclina/farmacología , Resultado del TratamientoRESUMEN
Poly(1-vinyl-2-pyrrolidinone) (PVP) and copolymers of 1-vinyl-2-pyrrolidinone are insoluble in water when crosslinked but they can absorb very large amounts of water to become syringe-injectable hydrogels. Such gels have been investigated recently as potential substitutes for the vitreous humour in the eye. In this study, during the cytotoxic evaluation by sulforhodamine B colorimetric assay of variously crosslinked PVP gels, it was found that many of them showed protective/growth promoting effects on 3T3 mouse fibroblasts in static cultures, a phenomenon encountered previously only with aqueous solutions of a limited number of natural or synthetic polymers. Particularly, the gels crosslinked with diethylene glycol dimethacrylate (DEGDMA) induced a significant enhancement of cell proliferation, especially in serum-free cultures. No correlation between this effect and the essential gel properties (chemical composition, viscoelasticity and equilibrium water content) could be established. The study demonstrated that crosslinked PVP hydrogels showed a serum-like growth promoting effect on an anchorage-dependent cell line, which may be due to physical protection, inability of the insoluble gels to penetrate cell membranes, and their ability to mimic the extracellular matrix.