Acrylic Acid Surface-Modified Contact Lens for the Culture of Limbal Stem Cells

Abstract

Surface treatment to a biomaterial surface has been shown to modify and help cell growth. Our aim was to determine the best surface-modified system for the treatment of limbal stem cell deficiency (LSCD), which would facilitate expansion of autologous limbal epithelial cells, while maintaining cultivated epithelial cells in a less differentiated state. Commercially available contact lenses (CLs) were variously surface modified by plasma polymerization with ratios of acrylic acid to octadiene tested at 100% acrylic acid, 50:50% acrylic acid:octadiene, and 100% octadiene to produce high-, mid-, and no-acid. X-ray photoelectron spectroscopy was used to analyze the chemical composition of the plasma polymer deposited layer. Limbal explants cultured on high acid-modified CLs outgrew more cells. Immunofluorescence and RT²-PCR array results indicated that a higher acrylic acid content can also help maintain progenitor cells during ex vivo expansion of epithelial cells. This study provides the first evidence for the ability of high acid-modified CLs to preserve the stemness and to be used as substrates for the culture of limbal cells in the treatment of LSCD.

Introduction

The integrity of the cornea, the most anterior part of the eye, is indispensable for vision. Forty-five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of loss of corneal transparency.¹ Limbal epithelial stem cells (LSCs) are a population of stem cells responsible for the maintenance and repair of the corneal surface.² Limbal stem cell deficiency (LSCD), in which pathologic insults destroy the stem cell-containing limbal epithelium or change the stem cell niche, can lead to vision impairment. LSCD can only be treated with limbal epithelial cell transplantation. Transplantation of ex vivo expanded LSCs, in which only a small biopsy (1–2 mm) is needed, was originally described by Pellegrini et al.³ This technique has the ability to restore the structural and functional integrity of the corneal surface and has now become a treatment option for patients with LSCD in several clinics.^4,5

The success rate of ex vivo expanded LSC transplantation depends on a variety of factors, including indication for surgery, the state of the ocular surface and lids.⁶ The adequate number of stem cells in the graft is an important factor in deriving a successful clinical outcome. Cultures containing more than 3% p63 (stem cell marker)-positive cells were found to be associated with successful transplantation in 78% of patients, whereas cultures containing 3% or less p63-positive cells were associated with only 11% of successful transplants.⁶ Several methods have been used to maintain the stem cell qualities of limbal cells and enhance proliferation.

To preserve the limbal epithelial cells in their stem-like stage, a feeder layer of growth-arrested mouse 3T3 fibroblasts was thought to be required. However, the support cells present a safety concern with the possibility of transmitting xenogeneic disease and incorporating or expressing immunogenic molecules present in animal products.⁷ Using the amniotic membrane (AM) as a substrate is the current gold standard method for the treatment of LSCD⁵; however, there are a number of drawbacks, such as considerable variation among donors, the risk of viral agent transmission, and ultimately, a lack of optimal transparency, which is obviously a major issue when dealing with repair of the cornea. Alternative materials that have been used for reconstruction of ocular surface include collagen, fibrin gel, and silk fibroin.⁸ He et al. have introduced collagen type I shields for corneal epithelium transplantation.⁹ Although encouraging results were recorded, high failure rates were observed that were possibly related to rapid resolution of the shields. Fibrin sealant, produced from combining fibrinogen and thrombin, has been shown to affect cells by causing differentiation although it has been used as a substrate for autologous epithelial limbal stem cell transplantation.^10,11 Silk fibroin has also been shown to support the growth of limbal epithelial cells; however, the cost of this natural material is considerably greater than the cost of synthetic materials.^12,13 Vitrigel membranes are another replacement for AM, but there is one major limitation for this procedure— cells cannot be seeded within the gel due to the lengthy (2 weeks) dehydration process.¹⁴ Due to the reasons stated above, few of these substrata have reached clinical trials or even experimentation in animal models.

Following the success of Myskin (a plasma polymer-coated surface) to support the transfer of autologous keratinocytes to patients with extensive burn injuries and chronic nonhealing diabetic ulcers, a similar approach using a contact lens (CL) system to help the transfer of cultured cells to the cornea for the treatment of LSCD has been developed. Recently, Di Girolamo et al. showed that the ocular surface epithelium can be expanded on a standard therapeutic CL, and subsequently described a novel and innovative method to treat LSCD.¹⁵ Other groups have used modified CL polymers to successfully engraft and track transplanted corneal epithelia in devitalized organ cultured pig and rabbit corneas.¹⁶ The CL method may be an ideal approach for culturing and transferring LSCs that does not depend on either the use of any xenogeneic cells or biologically variable donor material. Advances in conjugation chemistries have now widened the options for modifying synthetic biomaterials, including CL. As an adequate number of stem cells in the graft are closely related to a successful clinical outcome, it is worth knowing if CL can preserve the stemness of cultured limbal cells. The aim of this study was to demonstrate the ability of a modified CL system to maintain cultivated epithelial cells in a stem cell state.

Materials and Methods

Preparation of plasma copolymer-coated surfaces

The plasma reactor consists of a 2-L cylindrical sample chamber with an external wire coil and the sample chamber was kept under vacuum using a rotary pump. Plasma was sustained by a radio frequency (13.56 MHz) generator and was inductively coupled to the end flanges of the sample chamber. A schematic drawing of the plasma reactor is shown in Figure 1A. The base pressure within the reactor was typically within the range of 1.0×10⁻³–3.0×10⁻³ mbar.

FIG. 1.

(A) Schematic drawing of the plasma reactor. (B) contact lens (CLs) spectra of acrylic acid and curve fitted peaks. Area under the -COOH fitted curve was used to calculate the percentage of functional -COOH groups present on the surface. Table shows percentage of acid groups calculated for each surface type (n=9).

The precursor monomer acrylic acid (Sigma Aldrich) and 1,7-octadiene (Merck) were processed with three freeze–thaw cycles to remove any dissolved gas. The CLs were placed concave side facing upward within the chamber on a culture plate as a support base. A cleaned glass coverslip was also inserted for chemical characterization of the plasma polymer by X-ray photoelectron spectroscopy (XPS). The monomers were introduced into the chamber using a fine needle valve at various ratios, but always with a combined flow rate of 2 standard cubic centimeters per minute (sccm). Monomer ratios of acrylic acid to octadiene used to prepare the surfaces were 100% acid, 50:50, and 100% octadiene to produce high acid, low acid, and octodiene surfaces. Plasma was sustained at a power of 2 W over a period of 15 min.

For chemical characterization, a SPECS SAGE XPS system with a Phoibos 150 hemispherical analyzer and a MCD-9 detector was used. Nonmonochromated MgKα (hν 1253.6 eV), operated at 10 kV and 20 mA (200 W), was used. Survey spectra (0–1000 eV binding energy) were collected at a pass energy of 100 eV with a step size of 0.5 eV. To determine the chemical functionality of the deposited layer, narrow scans of the C 1s core level peak were collected at a pass energy of 20 eV with a step size of 0.1 eV. The relative amounts of -COOH present were identified for each sample from the C 1s narrow scan (Fig. 1). The spectra were analyzed with CASAXPS software (Neil Fairley) using relative sensitivity factors provided by SPECS. For curve fitting of the narrow scan C 1s core level peaks, a FWHM of 1.55 eV was used throughout. To compensate for surface charging, all binding energies were referenced to the C 1s aliphatic carbon peak at 285 eV.

Primary human limbal epithelial cell isolation and culture

Research-consented corneas were supplied by the Lions Eye Bank of Victoria. All experiments were conducted with ethical approval for the use of human tissue from the Royal Victorian Eye and Ear Hospital Human and Research Ethics Committee.

Cadaver corneoscleral button was excised from fresh globes (donor age ranging between 50 and 75 years), which had no previous ocular surgery, trauma, or disease, and procured within 5 h after death. Human donor corneas were stored in organ culture media (Eagle's MEM with 2% fetal bovine serum, antibiotics/antimycotics [Invitrogen] and L-glutamate [Glutamax] 0.29 mg/mL). Corneas were transferred to a transport medium (organ culture medium with 5% w/v dextran [mw 500,000]) 24 h before keratoplasty. After standard full-thickness corneal transplants (keratoplasty), remnant corneal rims were stored in the transport medium at room temperature.

We used the two established methods of culturing corneal epithelium: by cell suspension or through explants. Full-thickness limbal explants were prepared under a laminar flow. The Descemet's membrane was removed first and a 2 mm biopsy punch was used to excise explants to make each explant exactly the same size. For cell suspension, we followed the method described previously.¹⁵ Briefly, the limbal rim was incubated with dispase II (5 mg/mL) (Sigma) at 37°C for 1 h. The limbal epithelial sheets were collected and treated with 0.25% trypsin/0.03% EDTA at 37°C for 10 min to isolate single cells.

Culture of cells on plasma-coated CLs

CLs arrived dehydrated and were rehydrated overnight in the corneal culture medium. A single explant was placed epithelial side down on the center of each CL and placed in an incubator at 37°C for 30 min to facilitate their attachment to the lens. Fifty microliters of corneal medium was placed over each explant, and then the explants were returned to the incubator for 3 h, before being flooded with 4 mL of complete corneal medium. This prevented detachment from the CL following the flooding of the culture well. All culture dishes were then placed in an incubator at 37°C with 5% carbon dioxide and 95% air, and the growth medium was changed every second day.

Normally, more than 12 explants could be generated from a single limbal ring with the 2 mm biopsy punch (Fig. 2). The explants used for each experiment were sourced from one donor limbal ring to minimize the number of external factors distorting comparisons of lens performance. The distribution of stem cells may not be even throughout the limbus; there is evidence to suggest that they are predominately located in the superior and inferior quadrants of the limbus, with fewer located in the nasal and lateral quadrants.¹⁷ The number of viable stem cells is likely to be greater in explants derived from the superior and inferior limbus and this may lead to more rapid growth on the underlying lenses. To avoid this, explants from the same quarter of the ring were randomly distributed on different lenses. The quantification was drawn from 6 biological replicates (n=6), and mean values were used for further calculations.

FIG. 2.

(A) Limbal rings for corneal explants. Normally more than 12 explants could be generated from a single limbal ring, each measuring 2 mm in diameter. (B) Corneal explant on coated CLs. A single explant was placed epithelial side down on the center of each CL. CLs, contact lenses; AA, acrylic acid; OCT, 1,7-octadiene. Color images available online at www-liebertpub-com.web.bisu.edu.cn/tea

In the cell suspension method, the isolated limbal epithelial single cells were cultured at a density of 1.0×10⁵ cells per lens. They were cultured on the CLs and in a humidified 5% CO₂ incubator at 37°C, in 12-well plates. The media were changed every 2–3 days. Confluent cultures (10–14 days) were used for PCR array.

Assessment of cell viability

Cell attachment and viability on a range of plasma polymer surfaces were assessed using the resazurin metabolism assay. Resazurin is a phenoxazin-3-one dye known to act as an intermediate electron acceptor in the electron transport chain between the final reduction of oxygen and cytochrome oxidase by substituting for molecular oxygen.¹⁸ The deoxygenated product of resazurin, resorufin, exhibits strong emission at wavelengths greater than 550 nm. The resazurin metabolism test has been used since the 1950's and it has been commercialized since 1993 as Alamar Blue dye.¹⁹

Resazurin was dissolved in phosphate-buffered saline (PBS; 880 μM), filter sterilized, and stored in the dark at 4°C. For the assay, all media were removed from the culture well and 1950 μL of fresh corneal medium and then 50 μL of stock resazurin were added. A no cell control was prepared at the same time. Cultures were incubated for 6 h at 37°C and 5% CO₂. The medium was collected from each well and absorbance at 570 nm was determined with a Nanodrop spectrophotometer (Thermo Fisher Scientific) blanked against a no cell control. A standard curve formula derived from serial dilutions was used to determine cell numbers.¹⁹

Morphometric analysis

After 10 days, cells grown on CLs were fixed with a 4% paraformaldehyde solution for 10 min at room temperature. For each CL, images of different magnification were acquired for computerized analysis. For each image, ImageJ software was used to study morphology after individual contours were designated manually. The mean values of cell area were assessed for each CL.

Immunocytochemistry

Two putative stem cell markers were used to determine the degree of differentiation undertaken by cells radiating out from each explant. Several recent reports indicate that the p63 gene is essential for epithelial stem cell maintenance and differentiation.^20–22 The G2 member of the ATP binding cassette family (ABCG2) is regarded as a more specific marker for limbal stem cells.^23–25 On staining, the cells were washed three times with PBS, fixed for 10 min at room temperature in 2% (wt/vol) paraformaldehyde, and storage at −20°C. The cultures were blocked for 1 h in PBS supplemented with 5% goat serum and 0.5% Triton X followed by the primary antibodies (mouse monoclonal antibody for ABCG2 [ab24115; Abcam] and mouse monoclonal antibody against p63 protein [4A4; Santa Cruz]), respectively. Subsequently, the cells were incubated with their respective secondary antibodies (goat anti-mouse streptavidin-FITC and streptavidin-Alexa Fluor 633). Finally, cells were counterstained using 4,6-diamidino-2-phenylindole (DAPI) (Vector Labs). All incubations apart from the primary antibody incubation were performed at room temperature, and each step was interspersed with three 5-min rinses with PBS containing 0.1% Tween-20 (Sigma-Aldrich).

Normal corneas were used as control tissues. Immunofluorescent staining was performed by a previously reported method.²⁴ In brief, corneal and limbal sections were fixed with 2% paraformaldehyde, and after blocking with 5% normal goat serum in PBS for 30 min, the primary antibody and the respective secondary antibody were applied.

Human stem cell signaling RT² Profiler™ PCR array

The stem cell properties of the cells on high acid-coated CLs were compared with the cells on low acid-coated CLs by means of stem cell RT² Profiler™ PCR Array system (SA Biosciences). Total RNA was extracted from cells using Tri Reagent (Invitrogen) and template cDNA was synthesized using the SuperArray RT² First Strand Kit (SABiosciences) following the manufacturer's protocol. The template cDNA was mixed with 2×RT² qPCR Master Mix and aliquoted into each well on the PCR array plate containing predispensed gene-specific primer sets. Each sample was tested in triplicate by three repeated PCR array kits. Reactions were carried out using an ABI Prism 7300 Sequence Detection System (Applied Biosystems). PCR amplification followed a two-step cycling program: 10 min denaturizing at 95°C, 40 cycles of 95°C for 15 s, and 60°C for 1 min.

The expression of 5 housekeeping genes, 3 RNAs, and PCR quality controls, and 84 human genes related to stem cell-specific markers, stem cell differentiation markers, and signaling pathways important for stem cell maintenance were studied. Analysis of the expression of the panel of genes was determined by the defined template developed by the SABioscience Company following the instructions from the SuperArray Website (www.superarray.com=pcrarraydataanalysis.php).

Statistics

Differences between the groups were statistically evaluated using the one-way ANOVA or Student's t-test by the SPSS program and graphical analysis was made with Excel (Microsoft). The results are presented as mean±SEM. All p-values were two tailed, and a p-value of less than 0.05 was considered to be statistically significant.

Results

XPS characterization of plasma polymer surfaces

The successful deposition of carboxylic acid (-COOH) groups to the surface of the CL was confirmed using XPS and deconvolution of the narrow scan carbon peak (C 1s). In samples that were not plasma polymerized with acrylic acid, an acid peak in the CLs was not detected. We found that after 15 min of plasma deposition time, the chemistry of the underlying CL material was no longer evident, indicating that the plasma layer was sufficiently thicker than the XPS analysis depth of 10 nm. As the ratio of acrylic acid to octadiene changed, we saw a decrease in the acid peak in correlation with an increase in octadiene flow rate (Fig. 1B). The 100% octadiene surface majorly consists of hydrocarbon (-CH) groups (data not shown).

Ability of acrylic acid–coated lenses to support human epithelial cell culture

Cell growth was closely linked to adhesion of explants to the bottom of the culture well. Limbal human epithelial explants did not attach to octadiene only coated CLs, and thus, there was no cell layer grown on this type of CLs. A continuous monolayer of polygonal epithelial cells was seen on acrylic acid-coated CLs after 10 days in culture as shown by microscopy and hematoxylin–eosin staining. For cells grown on CLs, generally, there were more cells outgrown on high acid surface CLs than on low acid surface CLs and uncoated CLs (Fig. 3).

FIG. 3.

Corneal explants on contact lenses. (A) Limbal epithelial cells migrating from tissue explants on day 10 on different CLs. The central part was the corneal explant generated from the limbal ring with the 2 mm biopsy punch. (B) Light microscopy shows a single layer of human limbal epithelial cells outgrown on CLs. Arrows show the epithelial cell layer on the transparent CL. (C) Number of cells grown on different CLs using the resazurin metabolism assay (error bars±SE, n=6). Color images available online at www-liebertpub-com.web.bisu.edu.cn/tea

Immunocytochemistry

Immunofluorescent staining of histologic sections of human cornea and limbus showed that the ABCG2 transporter was expressed in the membrane and cytoplasm of certain basal cells in the human limbal epithelia, but not in the suprabasal layers of the limbal epithelium or in any layer of the corneal epithelium (Fig. 4A–D). p63 was expressed in the nuclei of certain basal cells in the human limbal epithelia (Fig. 4E).

FIG. 4.

Immunofluorescent and immunohistochemistry staining of histologic sections of the human cornea and limbus. Immunofluorescent staining of histologic sections of the human cornea and limbus shows that the ABCG2 transporter was expressed in the membrane and cytoplasm of certain basal cells in the human limbal epithelia, but not in the suprabasal layers of the limbal epithelium or in any layer of the corneal epithelium (B, D). Immunohistochemistry showed that p63 was expressed in the nuclei of certain basal cells in the human limbal epithelia (E). DAPI staining (A, C). Initial magnification ×10. Arrows show the p63 positive cells. DAPI, 4,6-diamidino-2-phenylindole. Color images available online at www-liebertpub-com.web.bisu.edu.cn/tea

There were different staining patterns on different CLs as well as in different parts of the outgrown on the same CLs. Consistently throughout the experiments in the cultured limbal explants, both p63 and ABCG2 were highly expressed in the small cells in the front edge. Only part of large flattened cells in the central part of the explants were p63 and ABCG2 positive (Fig. 5). About 94.23%±10% cells outgrown from the explants on high acid surface CLs were ABCG2 positive, while there were 88.15%±16% ABCG2-positive cells for low acid surface CLs (p=0.009) and 62.50%±11% for uncoated CLs. p63 was expressed by 64.58%±20% cells on high acid surface CLs, 40.36%±7% cells on low acid surface CLs, and only 26.11%±8% cells on uncoated CLs, respectively (p=0.48; Fig. 6).

FIG. 5.

Immunocytochemistry of cells outgrown on coated or uncoated CLs after cultured for 10 days. (A–F) Hundred percent acrylic acid-coated CLs. (A–C) Edge of the cells outgrown from the explant on 100% acrylic acid-coated CLs. (D–F) Central cells outgrown from the explant on 100% acrylic acid-coated CLs. (G–L) 50%:50% acrylic acid and octadiene-coated CLs. (G–I) Edge of the cells outgrown from the explant on 50% acrylic acid-coated CLs. (J–L) Central cells outgrown from the explant on 50% acrylic acid-coated CLs. (M–O) Uncoated lenses. ABCG2, green; p63, red; DAPI, blue. Initial magnification ×10. Color images available online at www-liebertpub-com.web.bisu.edu.cn/tea

FIG. 6.

Different staining patterns on different CLs. (A) p63 staining results (B) ABCG2 staining results.

Human stem cell signaling RT² Profiler PCR array

To confirm whether different acrylic acid compositions will influence the gene expression, we compared the gene expression of cells grown on different CLs. The change of over fourfolds was recognized as upregulation. The dissociation curve was analyzed for the 84 genes studied, and no DNA contamination was detected. The results indicated increased expression of nine genes and decreased expression of one gene for high acid surface CLs compared with low acid surface CLs (Table 1). Among the nine upregulated genes, six (ACVR1C, AMHR2, LTBP2, TGFBR1, EP300, and FGFR2) coded for the TGFβ superfamily signaling pathway and two (FZD4 and FZD9) for the Wnt signaling pathway. RGM family member A (RGMA) mRNA was downregulated.

Table 1.

RT²-PCR Array Results from Comparison of the Cells Grown on 100% Acrylic Acid Lens and 50% Acrylic Acid Lens Show Increased Expression of Nine Genes

TGFβ superfamily signaling pathway
Receptors & coreceptors	ACVR1C, AMHR2, LTBP2, FGFR2
Transcription factors & cofactors	EP300
Wnt signaling pathway
Receptors	FZD4. FZD9
Transcription factors & cofactors	TCF7L1
Notch signaling pathway
Receptors & coreceptors	NOTCH4

This figure shows a part of the Human Stem Cell Signaling PCR array analysis, including over fourfold upregulation of genes.

Discussion

The purpose of transplanting amplified cell sheets in the treatment of LSCD is to replace the defective epithelium with self-renewing stem cells, capable of maintaining a stable cornea. Therefore, preserving stem cells is vital in ex vivo limbal stem cell transplantation. We compared the growth characteristics of cells growing on CLs coated with different charged groups (acrylic acid and octadiene), and also the stem cell marker expression as well as the expression profile of 84 key genes involved in signal transduction pathways important for stem cell maintenance and differentiation. Our goal was to find out the optimal modified surface CLs for a corneal stem cell culture and transfer system. Such a system has several advantages as it eliminates any need for sutures, offers a stable supply, acts as a protective shield to facilitate healing and surface reconstruction, and may be an alternative to the use of the AM for patients with corneal LSCD. For the first time, we showed that high acid surface-modified CLs enhance ex vivo limbal stem cell stemness by upregulating stem cell gene expression, increasing proliferation ability, thus providing an ideal substratum for adhesion, migration, and rapid expansion of limbal epithelial cells from explants.

A higher proliferation with increasing acrylic acid was indicated, as shown by most cells being grown on high acid surface lenses in Figure 3. The low acid surface lens also demonstrated the ability to promote cell growth when compared with the uncoated CLs. Previous work has also demonstrated that there were differences noted in sustaining epithelial cell adhesion and growth on different CLs.²⁶ The results may be explained by the change of the hydrophobicity and surface-free energy after surface modification. Many studies have shown that hydrophobicity, surface energy, and charge of the material surface greatly influence the cell attachment and cell growth on the material.²⁷ The cell attachment belongs to the first phase of cell/material interactions and the quality of this phase will influence the cell's capacity to proliferate and to differentiate itself on contact with the material.²⁸ As shown in Figure 2, high acid surface CLs were more hydrophilic than the others, and a monomolecular layer of water is spontaneously formed on the surface.

A variety of stem cell markers have been proposed to identify limbal stem cells. The transcription factor p63 gene is a homologue of the tumor suppressor p53, and the first gene product definitely distinguishing stem cells from their transient amplifying (TA) progeny in stratified squamous epithelia.²⁹ p63 is critical for maintaining the progenitor cell populations that are necessary to sustain epithelial development and morphogenesis.³⁰ p63 is specifically expressed by stem cells of human epidermis and limbal epithelium, and not by TA cells. We have shown that p63 was highly expressed in the basal layers of many epithelial cells, as reported by other researchers.³¹ ABCG2 is a member of the ABC family of cell surface transport proteins, which mediates the transfer of a diverse array of substrates across cellular membranes. ABCG2 expression is relatively limited to primitive stem cells.²³ ABCG2 expression has been identified as a molecular determinant for the side population (SP) phenotype.²⁴ SP is highly enriched for hematopoietic stem cells with long-term repopulation activity after transplantation.³² Furthermore, ABCG2 has been proposed as a universal marker for stem cells from a variety of sources.³³ We have shown that the ABCG2 transporter was exclusively expressed by the limbal epithelium on the ocular surface and confirmed the exclusive expression pattern of the ABCG2 transporter, which is consistent with its expression and functional role in corneal epithelial cells that have already been reported.^10,24

Immunofluorescent staining for putative corneal epithelial stem cell markers, ABCG2 and p63, has been used in this study to compare the degree of differentiation of cells growing in different conditions. Figure 5 shows that ABCG2- and p63-positive cells were present, in varying numbers, throughout different regions of the cell sheet on coated acrylic acid lenses, with a vast number of positive cells observed on the high acid surface CLs. This suggests that the acrylic acid coating not only enhanced the corneal limbal cell ability to proliferate, but also contributed to the maintenance of their stem cell phenotype. Clinical results show that cultures containing more p63-bright cells were found to be associated with more successful transplantation in patients.⁶ Kawakita et al. have proved that p63-positive epithelial cell clusters had higher growth potential during ex vivo expansion.³⁴

The presence of both ABCG2- and p63-positive cells may indicate that the cells remain in a transitional phase in culture, somewhat between a stem cell and early TA cell. Previous work has shown that both rabbit and human limbal cells grown on TCP appear to be more differentiated than the cells on the acrylic plasma surface.¹⁶ It is likely that specific internal mechanisms also play an important role in this control process. To confirm this, we analyzed the gene expression. We chose the Human Stem Cell Signaling RT² Profiler PCR Array (PAHS047), which profiles the expression of 84 key genes involved in signal transduction pathways important for embryonic stem cell and induced pluripotent stem cell maintenance and differentiation. The array represents the genes for the receptors and transcription factors of the major signaling pathways involved in pluripotent cell maintenance and differentiation, including fibroblast growth factors, Hedgehog, Notch, TGFβ, and WNT.

Analysis of our RT²-PCR-A data indicated that nine genes increased expression when comparing cells grown on high acid surface CLs and low acid surface CLs (Table 1). Among the upregulated genes, FZD4 and FZD9 belong to the Frizzled family. As currently understood, the Wingless-MMTV Integration Site (Wnt) signaling proteins bind to receptors of the Frizzled and LRP families on the cell surface. Wnt signaling has been implicated in the control of various types of stem cells and may act as a niche factor to maintain stem cells in a self-renewing state.³⁵ Recently, Wnt signaling has been proven to be present in the ocular surface epithelium and play an important role in the regulation of LSC proliferation.³⁶ NOTCH4 was also upregulated in this study. The highly conserved Notch gene family plays an important role in cell fate decisions in a wide range of lineages in invertebrates and vertebrates. Recent work has shown that Notch4-IC, like Notch1-IC, plays a role in hematopoiesis, in that, it maintains stem cells and suppresses myeloid differentiation.³⁷ Other upregulated genes belong to the TGFβ superfamily signaling pathway, which is involved in cell differentiation. In general, Wnt and notch signaling pathways act to maintain the undifferentiated state of stem cells, while other growth factors instruct the cells to proliferate.

It is well known that the behavior of cells can be regulated by biochemical and topographical cues.³⁸ The goal in biomaterial surface modification is to influence the interactions between the material and the biological environment. Surface modification is a widely adopted method because it can enhance the biocompatibility of a material surface, while keeping the bulk properties intact. Among the different modification methods, the most common approach to polymer surface modification is coating of materials having appropriate biological properties through physical adsorption.³⁹ In surface modification of polymers, the carboxyl group is one of the best functional groups, being introduced to the surface with a covalent bond between the grafted layer and the substrate. Acrylic acid is commonly used in such grafting processes, which act as a spacer to bind proteins and the substrate.⁴⁰ We have proved that high acid surface-modified CLs represent a good alternative to commercial CLs for both growing corneal cells and maintaining the characteristics of stem cells. Introducing this methodology will improve convenience for the clinic, and a clinical trial is being undertaken in a collaborating hospital. Furthermore, the functional analysis of our results has provided a better understanding of the molecular signaling pathways associated with surface coating. This knowledge could have substantial benefits for the future of regenerative medicine, biomaterials, and tissue engineering.

Footnotes

Acknowledgments

The Australia-India Strategic Research Fund BF020074 and the National Natural Science Foundation of China 81201184. (“An Advanced Surface for the Cell Therapy of Limbal Epithelium for Ocular Surface Disease: Proof of Concept and Clinical Trials”).

Disclosure Statement

No competing financial interests exist.

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Acrylic Acid Surface-Modified Contact Lens for the Culture of Limbal Stem Cells

Abstract

Introduction

Materials and Methods

Preparation of plasma copolymer-coated surfaces

Primary human limbal epithelial cell isolation and culture

Culture of cells on plasma-coated CLs

Assessment of cell viability

Morphometric analysis

Immunocytochemistry

Human stem cell signaling RT 2 Profiler™ PCR array

Statistics

Results

XPS characterization of plasma polymer surfaces

Ability of acrylic acid–coated lenses to support human epithelial cell culture

Immunocytochemistry

Human stem cell signaling RT 2 Profiler PCR array

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Human stem cell signaling RT² Profiler™ PCR array

Human stem cell signaling RT² Profiler PCR array