A high-density PEG interfacial layer alters the response to an EGF tethered polydimethylsiloxane surface

Previously, epidermal growth factor (EGF)-modified surfaces have shown promise in supporting cellular growth and adhesion on synthetic polymeric substrates. Surfaces prepared using a novel modification technique were investigated in the current work for their ability to support corneal epithelialization, important to the integration of a synthetic artificial cornea. EGF could be tethered to PDMS surfaces via a high-density, hetero-bifunctional PEG-NSC linking layer with a tunable surface concentration of up to 300 ng/cm(2). Only a small fraction of the EGF on these surfaces could be removed with SDS rinsing, indicative of covalent tethering. Studies with human corneal epithelial cells suggest a relatively linear increase in the number of corneal epithelial cells with increasing EGF concentration at all times. However, confluence was not achieved at any time point. It is believed that the presence of the non-adsorbent PEG layer, useful for preventing non-specific adsorption of proteins, may limit the cellular response by minimizing the adsorption of adhesion molecules. The effects of the EGF alone are clearly not sufficient to result in epithelialization of an artificial cornea surface. Altering both the adhesion and growth of corneal epithelial cells in a controlled manner may be necessary for epithelialization of an artificial cornea.     

EGF-grafted PDMS surfaces in artificial cornea applications

Lack of epithelial cell coverage has remained a persistent problem in the design of an artificial cornea. In this work, polydimethylsiloxane (PDMS) surfaces were modified with epidermal growth factor (EGF) to improve the growth of corneal epithelial cells. The EGF was covalently tethered to PDMS substrates aminated by plasma polymerization of allylamine via a homobifunctional polyethylene glycol (PEG) spacer. Surface modification was confirmed by contact angle and X-ray photoelectron spectroscopy measurements. By varying the ratio of EGF to PEG from 1:50 to 1:5, EGF amounts from 40 to 90 ng/cm2 could be bound, as determined by surface plasmon resonance (SPR) and 125I radiolabelling. Human corneal epithelial cells on the various modified surfaces were cultured both in the presence and absence of EGF in the culture medium to determine the effect of covalently bound EGF on the cells. The results demonstrated that covalently bound EGF on the surfaces is active with respect to promoting epithelial cell coverage. This was significant when compared to unmodified controls.     

Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium

Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.     

Interactions of corneal cells with transforming growth factor beta 2-modified poly dimethyl siloxane surfaces

The downgrowth of corneal epithelial cells at the interface of an artificial cornea and the host eye tissue poses a significant problem to be overcome in developing a successful implant. As a means of inhibiting the proliferation of corneal epithelial cells on the stromal surface of the implant, we examined the immobilization of transforming growth factor beta-2 (TGF-beta2) via a bifunctional poly ethylene glycol (PEG) spacer to poly dimethyl siloxane (PDMS) surfaces. Growth factor immobilization was confirmed by modification with (125)I-labeled TGF-beta 2. The modified surfaces were also characterized by advancing water contact angles, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Although the amount of growth factor covalently bound to the surface was difficult to quantify apparently due to strong interactions between the growth factor and the PEG layer and high levels of adsorption, differences in the modified surfaces, suggestive of the presence of a significant amount of TGF-beta 2, were found. In vitro interactions of the modified surfaces with human corneal epithelial and stromal cells were examined. Growth factor surface concentrations as well as culture in the absence and presence of serum and other adhesive proteins were examined. Corneal stromal and epithelial cells cultured on the TGF-beta 2-modified surfaces consistently gave results opposite to those expected. Likely, the most notable and surprising result was the almost complete lack of adhesion of the stromal cells, with coverage averaging between 3 and 5%. In comparison, corneal epithelial cell growth appeared to be promoted by the presence of the immobilized growth factor, with cell coverage averaging 50-60% at 7 days of culture. A TGF-beta 2 concentration effect was noted with both cell types in the absence of serum, with increases in the coverage at higher TGF-beta 2 concentrations. The observed cell growth appeared to be the result of interactions between the cells and active growth factor, because the addition of anti-TGF-beta 2 to the culture medium reduced cell coverage to levels similar to those noted on control surfaces. Therefore, although TGF-beta 2-modified surfaces may not be suitable as corneal epithelial cell inhibiting surfaces, interactions of surface immobilized growth factor and corneal cells are complex and should be further examined.     

Design and fabrication of an artificial cornea based on a photolithographically patterned hydrogel construct

We describe the design and fabrication of an artificial cornea based on a photolithographically patterned hydrogel construct, and demonstrate the adhesion of corneal epithelial and fibroblast cells to its central and peripheral components, respectively. The design consists of a central “core” optical component and a peripheral tissue-integrable “skirt.” The core is composed of a poly(ethylene glycol)/poly(acrylic acid) (PEG/PAA) double-network with high strength, high water content, and collagen type I tethered to its surface. Interpenetrating the periphery of the core is a microperforated, but resilient poly(hydroxyethyl acrylate) (PHEA) hydrogel skirt that is also surface-modified with collagen type I. The well-defined microperforations in the peripheral component were created by photolithography using a mask with radially arranged chrome discs. Surface modification of both the core and skirt elements was accomplished through the use of a photoreactive, heterobifunctional crosslinker. Primary corneal epithelial cells were cultured onto modified and unmodified PEG/PAA hydrogels to evaluate whether the central optic material could support epithelialization. Primary corneal fibroblasts were seeded onto the PHEA hydrogels to evaluate whether the peripheral skirt material could support the adhesion of corneal stromal cells. Cell growth in both cases was shown to be contingent on the covalent tethering of collagen. Successful demonstration of cell growth on the two engineered components was followed by fabrication of core-skirt constructs in which the central optic and peripheral skirt were synthesized in sequence and joined by an interpenetrating diffusion zone.     

Cytocompatibility of Three Corneal Cell Types with Amniotic Membrane

Rabbit limbal corneal epithelial cells, corneal endothelial cells and keratocytes were cultured on amniotic membrane. Phase contrast microscope examination was performed daily. Histological and scan electron microscopic examinations were carried out to observe the growth, arrangement and adhesion of cultivated cells. Results showed that three corneal cell types seeded on amniotic membrane grew well and had normal cell morphology. Cultured cells attached firmly on the surface of amniotic membrane. Corneal epithelial cells showed singular layer or stratification. Cell boundaries were formed and tightly opposed. Corneal endothelial cells showed cobblestone or polygonal morphologic characteristics that appeared uniform in size. The cellular arrangement was compact. Keratocytes elongated and showed triangle or dendritic morphology with many intercellular joints which could form networks. In conclusion, amniotic membrane has good scaffold property, diffusion effect and compatibility with corneal cells. The basement membrane side of amniotic membrane facilitated the growth of corneal epithelial cells and endothelial cells and cell junctions were tightly developed. The spongy layer of amniotic membrane facilitated the growth of keratocytes and intercellular joints were rich. Amniotic membrane is an ideal biomaterial for layering tissue engineered cornea.     

Effects of insulin-like growth factor 2 and its receptor expressions on corneal repair

Limbal stem cell (LSC) on the basal layer of cornea plays an important role in the epithelial repair after corneal injury as it can proliferate, differentiate and migrate into injury sites under the direction of cytokines. This study explored the signaling pathway and cellular mechanism between corneal epithelial cells LSC, on a mouse model with mechanic corneal injury. Ipsilateral corneal mechanic injury model was prepared on mice using the contralateral eye as the control. Tissues from both central and peripheral regions of cornea were collected, cultured and quantified for expression of various cytokines including epidermal growth factor (EGF), fibroblast growth factor-β (FGF-β), heparin-like growth factor (HGF), keratinocyte growth factor (KGF), transforming growth factor-β1 (TGF-β1), IGF-1 and IGF-2. The effects of these factors on the differentiation of LSC and fibroblasts were also studied. Most of those cytokines had elevated gene expressions after the corneal injury. Among those IGF-2 had significantly increased expression, along with the high expression of IGF-2 receptor in corneal peripheral cells. IGF-2 also induced the differentiation of LSC into keratin-12-positive cells. Further studies showed the prominent expression of α-actin in injured tissues, suggesting the potential transformation of fibroblasts into myofibroblasts. Both IGF-2 and its receptor had elevated expressions after corneal injury. They may facilitate the transformation of LSC into epithelial cells, in addition to the role in transformation from fibroblasts to myofibroblasts.     

Comparison of corneal epithelial cellular growth on synthetic cornea materials.

The application of artificial corneas for severely wounded ocular surfaces has always encountered the problem of biocompatibility with corneal epithelial cells (CECs). For the eye to stay healthy, it must continually have a complete sheet of CECs across the artificial corneal surface. Various surface modifications of different polymeric materials have been examined to determine which have the best cellular growth rates. A mathematical model of corneal cell growth profiles on synthetic materials was formulated based upon a linear mitotic growth rate. Experimental data reported for the CEC growth on modified poly(vinyl alcohol), silicone rubber, polystyrene, and polycarbonate was analyzed using the model to estimate the linear mitotic rate constant (k). The model proved to be useful in comparing data from different investigators. Plasma-induced graft copolymerized poly(hydroxyethyl methacrylate) (pHEMA) on silicone rubber provided the best growth rate from this particular set of data.     

Modulation of corneal epithelial stratification by polymer surface topography.

The topography and porosity of a polymer may affect the epithelialization of a corneal implant. We used an in vitro model to examine the effect of polymer surface topography on corneal epithelial tissue stratification and the deposition of proteins associated with epithelial adhesion. A range of topographies was provided by polycarbonate membranes with nominal pore diameters of 0.1, 0.4, 0.8, 1.0, 2.0, or 3.0 microm and a nonporous surface. Stratification of epithelial tissue outgrowth on these surfaces was evaluated using light and electron microscopy. Deposition of proteins associated with basement membrane and adhesion complex formation at the tissue-polymer interface was assessed using immunohistochemistry. Surfaces with pores in the 0.1-0.8-microm-diameter range supported superior stratification and protein deposition compared with those containing pores of > or = 1.0 microm. Cytoplasmic processes penetrated single pores 2.0 and 3.0 microm in diameter and fused pores 1.0 microm in diameter. Tissue on the nonporous surface had a lower level of stratification compared with surfaces with pores 0.1-0.8 microm in diameter. These results point to the significance of surface topography in biomaterial applications that require persistent epithelialization.     

Adhesion of corneal epithelial cells to cell adhesion peptide modified pHEMA surfaces

Epithelialization of a corneal implant is a desirable property. In this study we compared surface modification of poly (2-hydroxyethyl methacrylate) (pHEMA) with the cell adhesion peptides RGDS and YIGSR. Various parameters in the tresyl chloride activation and modification reactions were considered in order to maximize surface coverage with the peptide including tresyl chloride reaction solvent. tresyl chloride reaction time, tresyl chloride concentration, peptide concentration, and peptide reaction pH. Surface chemistry and corneal epithelial cell adhesion to the modified surfaces were examined. X-ray photoelectron spectroscopy data suggested that while peptide modification had occurred, surface coverage with the peptide was incomplete. Acetone was found to result in a higher fraction of nitrogen and surface bound carboxyl groups compared to dioxane and ether. Furthermore, corneal epithelial cell adhesion to the surfaces for which acetone was used for the activation reaction was significantly greater. Statistical analysis of the various samples suggests that lower peptide concentrations and higher tresyl chloride reaction times result in better cell adhesion. Furthermore, modification with YIGSR resulted in higher surface concentrations and better cell adhesion than modification with RGDS. Little or no cell adhesion was noted on the unmodified pHEMA controls. Protein adsorption results suggest that the differences in cell adhesion cannot be attributed to differences in serum protein adsorption from the culture medium. We conclude that YIGSR modified surfaces have significant potential for further development in corneal applications.     

Amine functional hydrogels as selective substrates for corneal epithelialization

The aim of this study was to develop a synthetic hydrogel to act as a corneal substitute capable of selectively supporting the adhesion and proliferation of limbal epithelial cells (LECs) while inhibiting growth of limbal fibroblasts. Deficiency of LECs causes conjunctival epithelial cells to move over the cornea, producing a thick scar pannus. Unilateral defects can be treated using LEC cultured from the unaffected eye, transplanting them to the affected cornea after scar tissue is removed. The underlying wound bed is often damaged, however, hence the need to develop a corneal inlay to aid in corneal re-epithelialization. Transparent epoxy-functional polymethacrylate networks were synthesized using a combination of glycerol monomethacrylate, ethylene glycol dimethacrylate, lauryl methacrylate and glycidyl methacrylate that produced two different bulk hydrogel compositions with different equilibrium water contents (EWCs): Base 1 and Base 2, EWC = 55% and 35%, respectively. Two sets of amine-functional hydrogels were produced following reaction of the epoxide groups with excesses of either ammonia, 1,2-diamino ethane, 1,3-diamino propane, 1,4-diamino butane or 1,6-diamino hexane. Neither series of hydrogels supported the proliferation of limbal fibroblasts irrespective of amine functionalization but they both supported the adhesion and proliferation of limbal epithelial cells, particularly when functionalized with 1,4-diamino butane. With Base 1 hydrogels (less so with Base 2) a vigorous epithelial outgrowth was seen from small limbal explants and a confluent epithelial layer was achieved in vitro within 6 days. The data support the development of hydrogels specific for epithelial formation.     

脱细胞猪角膜表面基底膜成分的检测

背景：前期实验显示脱细胞猪角膜具有良好的组织相容性,可以支持角膜细胞和皮肤上皮细胞的生长。 目的：检测脱细胞猪角膜是否保存了利于角膜上皮细胞生长的重要组织结构—基底膜。 方法：利用荧光抗体对脱细胞猪角膜表面的基底膜成分(层粘蛋白和Ⅳ型胶原)进行免疫组织化学检测,荧光显微镜下观察脱细胞猪角膜表面是否保存了基底膜成分。 结果与结论：免疫荧光染色显示脱细胞猪角膜前基质表面层粘蛋白和Ⅳ型胶原呈阳性表达,与新鲜猪角膜表面基底膜的荧光表达相同,表明脱细胞猪角膜保存了利于角膜上皮细胞生长的基底膜。     

Corneal epithelial cell growth over tethered-protein/peptide surface-modified hydrogels

In this study, we investigated the corneal epithelial cell growth rate and adhesion to novel hydrogels with (1) extracellular matrix proteins [fibronectin, laminin, substance P, and insulin-like growth factor-1 (IGF-1)] and (2) peptide sequences [RGD and fibronectin adhesion-promoting peptide (FAP)] tethered to their surface on poly(ethylene glycol) (PEG) chains. The growth rate to confluence of primary rabbit cornea epithelial cells was compared for plain polymethacrylic acid-co-hydroxyethyl methacrylate (PHEMA/MAA) hydrogels, PHEMA/MAA hydrogels coated with extracellular matrix proteins or peptides, and PHEMA/MAA hydrogels with tethered extracellular matrix proteins or peptides on the surface. The development of focal adhesions by the epithelial cells grown on the surfaces was determined by F-actin staining. Little to no epithelial cell growth occurred on the plain hydrogel surfaces throughout the 15-day culture period. Of the coated hydrogels, only the fibronectin-coated surfaces showed a significant increase in cell growth compared to plain hydrogels (p < 0.009). However, even these surfaces reached a maximum of only 20% confluence. Laminin, fibronectin adhesion-promoting peptide (FAP), and fibronectin/laminin (1:1) tether-modified hydrogels all achieved 100% confluence by the end of the culture period, although the rates at which confluence was reached differed. F-actin staining showed that focal adhesions were formed for the laminin, FAP, and fibronectin/laminin tether-modified surfaces. The results support the hypothesis that tethering certain extracellular matrix proteins and/or peptides to the hydrogel surface enhances epithelial cell growth and adhesion, compared with that seen for protein-coated or plain hydrogel surfaces.     

小鼠角膜的发生与细胞凋亡

目的 通过观察小鼠角膜的发生过程,探讨角膜细胞的增殖与凋亡对角膜结构修复与塑形的作用。方法 各日龄共计120只小鼠,用HE染色或4’,6-二脒基-2-苯基吲哚（DAPI）染色对小鼠角膜的一般结构进行观察;用5′-溴脱氧尿嘧啶核苷（BrdU）技术标记角膜增殖细胞和免疫荧光法标记干细胞和凋亡细胞。结果 胚胎发育及出生后早期,角膜以实质层的发育为主。出生14d（P14）左右,角膜上皮细胞层开始增殖分化为两层细胞,同时内皮细胞也开始分化。至P30时,我们可以辨别出角膜的6层结构。BrdU阳性细胞主要存在实质层中的成纤维细胞,出生以后也可见于角膜上皮细胞层和内皮细胞层。随着角膜发育,P10左右,其他层的BrdU阳性细胞都消失,仅存在于角膜上皮细胞层。增殖细胞核抗原（PCNA）阳性细胞在发育早期散在分布于角膜的各层,P14以后PCNA阳性细胞均匀的分布于角膜上皮细胞的基底层,并维持在稳定状态。在角膜发育早期,在各层可见许多细胞凋亡。结论 角膜的发育与其感光功能形成的过程相一致,角膜干细胞的增殖与其修复有关;有大量的凋亡细胞参与角膜结构的塑形。     

Can bone marrow cells give rise to cornea epithelial cells?

The corneal epithelium plays a critical role in maintaining the cornea's transparency and its avascularity. Severe damage to the limbal region results in serious problems with the corneal surface such as persistent epithelial defects, conjunctivalisation with vascularisation, keratinisation, scarring, etc. with associated profound visual loss. In order to rescue such damaged ocular surfaces, corneal epithelial stem cells were used to reconstruct artificial corneas by employing tissue engineering method. This procedure, however, requires a large limbal graft from the healthy eye and it is not possible in patients who have bilateral lesions. Therefore we should find other autologous cells as a source of cells for the reconstruction of the corneal surface. c-kit+ enriched bone marrow stem cells can give rise to different epithelial cells. So we hypothesize that this might apply to the cornea as well. Cultured cell sheets composed of autologous c-kit+ enriched bone marrow stem cells may be used to reconstruct corneal surfaces and can restore vision in patients with bilateral severe disorders of the ocular surface.     

Heparin-modified dendrimer crosslinked collagen matrices for the delivery of heparin-binding epidermal growth factor

A tissue engineered corneal equivalent (TECEs) requires host integration to achieve adequate anchorage and long-term device stability. Corneal integration through epithelialization and stromal integration can be manipulated by growth factors. We investigated the potential of heparin-binding epidermal growth factor (HB-EGF) for mediating interactions with human corneal epithelial cells (HCEC) and compared its efficacy to epidermal growth factor (EGF) in vitro. Furthermore, we utilized heparinized dendrimer crosslinked collagen gels, intended for use as TECE, for delivery of HB-EGF in a sustained manner. HCEC were exposed to HB-EGF at varying concentrations between 0.1 and 1000 ng/mL. Cell proliferation increased with growth factor concentration up to a concentration of 50 ng/mL, suggesting growth factor receptor down-regulation at higher HB-EGF concentrations. Response to HB-EGF was comparable to EGF at low concentrations of 0.1 and 1 ng/mL but at a concentration of 10 ng/mL, HB-EGF induced significantly better proliferation than EGF. Proliferation was found to be dependent on the initial seeding density. Heparinized dendrimer crosslinked collagen (CHG) gels were capable of HB-EGF uptake, which was influenced by heparin concentration within the gel, growth factor concentration and exposure time to the growth factor. HB-EGF release followed first order kinetics, with ～90% of the growth factor released after 2 weeks. Growth factor stability was verified with in vitro HCEC culture studies. Bioavailability was maintained in the gels through heparin interaction. Overall, HB-EGF induced proliferation of HCEC in vitro and can be released from heparinized collagen gels making it potentially suitable for promoting epithelialization of TECEs.     

Collagen-immobilized poly(vinyl alcohol) as an artificial cornea scaffold that supports a stratified corneal epithelium

The cornea is a transparent tissue of the eye, which is responsible for the refraction of incoming light. Both biological corneal equivalents and synthetic keratoprostheses have been developed to replace donor tissue as a means to restore vision. However, both designs have drawbacks in terms of stability and biocompatibility. Clinically available synthetic devices do not support an intact epithelium, which poses a risk of microbial infection or protrusion of the prosthesis. In the present study, type I collagen was immobilized onto poly(vinyl alcohol) (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. Human and rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-COL. The epithelial sheet expressed keratin 3/12 differentiation markers, the tight junction protein occludin, and had characteristic microvilli structures on transmission electron microscopy. Functionally, the stratified epithelium contained normal glycogen levels, and an apical tight-junction network was observed to exclude the diffusion of horseradish peroxidase. Furthermore, the epithelium-PVA-COL composite was suturable in the rabbit cornea, suggesting the possibility of using PVA-COL as a biocompatible material for keratoprosthesis.     

The effect of in vivo grown corneal epithelium transplantation on persistent epithelial defects with limbal stem cell deficiency

We report our experience with corneal epithelium, grown in vivo, transplantation in three patients with persistent epithelial defect (PED). The three patients had ocular surface disease unresponsive to standard treatments and were therefore chosen for transplantation. They underwent transplantation of epithelial sheets, grown in vivo, to the most affected eye. In vivo cultivation was carried out in the cornea of a living related donor. After epithelialization was completed, the epithelium grown on an amniotic membrane was harvested gently; it was then transplanted into the patient's eye after debridement of fibrovascular tissue. The cultivated epithelium was completely epithelialized by 2 weeks; it was well-differentiated with well-formed hemidesmosome. On immunohistochemical staining, p63, connexin 43, and Integrin beta4 were expressed in the cells on the epithelial sheet. The PED was covered completely and maintained for 4 weeks in all cases. However, corneal erosion recurred after 5 weeks in two cases. This novel technique demonstrates the corneal epithelial cells can be expanded in vivo successfully on denuded amniotic membrane of a healthy cornea and harvested safely. A corneal epithelial sheet, grown in vivo, can be transplanted to treat eye with a severe ocular surface disease, such as total limbal deficiency.     

Interactions of corneal epithelial cells and surfaces modified with cell adhesion peptide combinations

In order to facilitate the adhesion of corneal epithelial cells to a poly dimethyl siloxane (PDMS) substrate ultimately for the development of a synthetic keratoprosthesis, PDMS surfaces were modified by covalent attachment of combinations of cell adhesion and synergistic peptides derived from laminin and fibronectin. Peptides studied included YIGSR and its synergistic peptide PDSGR from laminin and the fibronectin derived RGDS and PHSRN. Surfaces were modified with combinations of peptides determined by an experimental design. Peptide surface densities, measured using 125-I labeled tyrosine containing analogs, were on the order of pmol/cm2. Surface density varied as a linear function of peptide concentration in the reaction solution, and was different for the different peptides examined. The lowest surface density at all solution fractions was obtained with GYRGDS, while the highest density was consistently obtained with GYPDSGR. These results provide evidence that the surfaces were modified with multiple peptides. Water contact angles and XPS results provided additional evidence for differences in the chemical composition of the various surfaces. Significant differences in the adhesion of human corneal epithelial cells to the modified surfaces were noted. Statistical analysis of the experimental adhesion results suggested that solution concentration YIGSR, RGDS, and PHSRN as well as the interaction effect of YIGSR and PDSGR had a significant effect on cell interactions. Modification with multiple peptides resulted in greater adhesion than modification with single peptides only. Surface modification with a control peptide PPSRN in place of PHSRN resulted in a decrease in cell adhesion in virtually all cases. These results suggest that surface modification with appropriate combinations of cell adhesion peptides and synergistic peptides may result in improved cell surface interactions.     

Influence of the substrate binding characteristics of fibronectin on corneal epithelial cell outgrowth. Student Research Award in the Doctoral Degree Candidate Category, Fourth World Biomaterials Congress (18th annual meeting of the Society for Biomaterials), Berlin, Germany, April 24-28, 1992.

The outgrowth of corneal epithelial cells onto a polymeric substrate is expected to be the primary event in the epithelialization of a synthetic corneal graft. Circular corneal buttons (5 mm) were punched from excised rabbit corneas and placed onto bare substrates or substrates preadsorbed with fibronectin (fn), albumin, or binary mixtures of both fn and albumin. Cell outgrowth areas were measured after culturing the buttons for 4 days in serum-free medium. Fibronectin adsorption to the materials was measured from pure and binary solutions with 125I-radiolabeled fibronectin. A parameter thought to be related to the binding strength of fn to polymeric substrates was measured in parallel experiments by partial elution of the adsorbed fn by 3% sodium dodecyl sulfate (SDS). Following pure solution fibronectin adsorption a range of outgrowth areas was measured (from 0.86 +/- 0.03 cm2 for glass to 1.49 +/- 0.03 cm2 for TCPS). On all of the materials tested cell outgrowth areas increased following fn preadsorption and decreased following albumin preadsorption relative to bare surfaces (p less than 0.05). Following preadsorption with binary protein mixtures cell outgrowth areas increased with fibronectin adsorption, however, the outgrowth areas were not determined solely by the concentration of fn adsorbed onto the surfaces. This result suggested that the biological efficiency of the adsorbed fibronectin was substrate-dependent. When the cell outgrowth data were cross-plotted against fn retention following SDS elution, the outgrowth areas were found to increase along with increases in fn retention. Based on these data we suggest that epithelial cell outgrowth may be partially governed by the tightness of binding between the fn molecules and the underlying substrate.