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(Investigative Ophthalmology and Visual Science. 2006;47:105-112.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0512
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The Development of a Serum-Free Derived Bioengineered Conjunctival Epithelial Equivalent Using an Ultrathin Poly({varepsilon}-Caprolactone) Membrane Substrate

Leonard P. K. Ang,1,2,3 Zi Yuan Cheng,3 Roger W. Beuerman,2,3 Swee Hin Teoh,4 Xiao Zhu,3 and Donald T. H. Tan1,2,3

1From the Singapore National Eye Center, Singapore; the Departments of 2Ophthalmology and 4Mechanical Engineering, National University of Singapore, Singapore; and the 3Singapore Eye Research Institute, Singapore.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To evaluate the use of an ultrathin poly({epsilon}-caprolactone) (PCL) membrane as a substrate for the development of a serum-free–derived conjunctival epithelial equivalent.

METHODS. Ultrathin PCL membranes 6 µm in thickness were prepared by solvent casting and biaxial stretching and analyzed by atomic force microscopy (AFM), scanning electron microscopy (SEM), tensile testing, and water-contact angle measurement. Rabbit conjunctival epithelial cells were cultivated on sodium hydroxide (NaOH)-treated PCL membranes and untreated PCL membranes in serum-free medium. The proliferative capacity of cultivated cells was analyzed with a bromodeoxyuridine (BrdU) ELISA proliferation assay. Conjunctival equivalents were xenografted into severe combined immune-deficient (SCID) mice. Immunostaining for tissue-specific and basement membrane-related proteins was performed.

RESULTS. After biaxial stretching, the tensile strength of PCL membranes increased from 21 to 42 MPa, with a Young’s modulus of 225 MPa. AFM and SEM showed that biaxially stretched PCL membranes consisted of closely packed microfibrils. PCL membranes supported the attachment and proliferation of conjunctival epithelial cells to form confluent stratified epithelial sheets. Surface modification with NaOH resulted in greater hydrophilicity and cellular proliferation than that of untreated membranes. Transplanted conjunctival equivalents underwent greater proliferation and stratification in vivo. Cultivated conjunctival cells expressed K4, K19, MUC5AC, and Ki67, whereas collagen IV and integrin ß4 were detected at the basement membrane junction.

CONCLUSIONS. An ultrathin PCL membrane was shown to be biocompatible, mechanically strong enough to stand up to handling, and able to support conjunctival epithelial cell proliferation. This membrane may have potential for use as a scaffold matrix for tissue-engineered conjunctival equivalents.

Ocular surface diseases, such as Stevens-Johnson syndrome, chemical and thermal burns, and inflammatory lesions result in damage to the corneal, limbal, and conjunctival epithelia, often resulting in significant visual morbidity. Conjunctival damage may also be damaged iatrogenically through ophthalmic surgical procedures that require manipulation and removal of the conjunctiva, such as in pterygium surgery, glaucoma surgery, and oculoplastic procedures. Cultivated limbal stem cell transplantation has been used in the treatment of limbal stem cell deficiency.1 2 3 More recently, cultivated conjunctival epithelial transplantation has also been used effectively as a means of conjunctival replacement in a variety of ocular surface disorders.4 5 The substrates that have been used for the development of ocular surface tissue-equivalents include human amniotic membrane (HAM), collagen gel and fibrin glue, of which HAM is currently the most commonly used substrate for ocular surface transplantation.1 2 3 4 5 6 7 8 9 However, the use of allogeneic biological material is associated with the risks of disease transmission (e.g., HIV, hepatitis B and C, and bacterial and fungal infections) and allograft rejection. In addition, many of the substrates lack the mechanical properties that allow easy handling and suturing, as well as prolonged endurance after transplantation.

As such, there is a perceived need to develop new methods of ocular surface epithelial cell replacement. The use of biosynthetic materials as stromal substitutes to support epithelial cell growth would overcome some of the problems related to the use of allogeneic tissue and biological substrates. These materials may be custom fabricated to suit each condition and could provide a ready supply of material for clinical use. Synthetic bioresorbable polymeric materials have been used as matrices for dermal equivalents for skin regeneration.10 11 12 13 14 These include of a polyglactin mesh, poly-L-lactic acid, and a block copolymer of poly(ethylene glycolterephthalate) and poly(butylene terephthalate).15 16 These substrates were shown to support the proliferation of epidermal keratinocytes and fibroblasts. However, these matrices still have shortcomings, such as poor mechanical strength and risk of immunologic rejection.

Poly({varepsilon}-caprolactone) (PCL) is a U.S. Food and Drug Administration–approved bioresorbable, biocompatible polymer that has good mechanical properties when biaxially stretched.17 18 19 20 We investigated the novel use of a biaxially stretched, synthetic ultrathin PCL membrane in the development of a serum-free derived conjunctival epithelial equivalent, and compared it with the use of an amniotic membrane substrate.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Dulbecco’s modified Eagle’s medium (DMEM), human epidermal growth factor (hEGF), penicillin, streptomycin, amphotericin B, trypsin, and EDTA were purchased from Invitrogen-Gibco (Grand Island, NY); insulin, hydrocortisone, bovine pituitary extract, and cholera toxin from Sigma-Aldrich (St. Louis, MO); keratinocyte growth medium (KGM) from BioWhittaker (Walkersville, MD); fetal bovine serum (FBS) from Hyclone (Logan, Utah); and bromodeoxyuridine (BrdU) and anti-BrdU antibody from GE Healthcare (Freiburg, Germany).

Normal mouse monoclonal IgG antibody, mouse monoclonal IgG antibody to cytokeratin K4, and pancytokeratin (AE-1 and AE-3) were purchased from Sigma-Aldrich and antibodies to K19 and Ki67 from DakoCytomation (Carpinteria, CA). AE-5 (antibody to K3) was a kind gift from Tung-Tien Sun (New York University, New York, NY). Mouse monoclonal antibody to MUC5AC was purchased from Chemicon (Temecula, CA). Biotinylated horse anti-mouse immunoglobulin G was obtained as a peroxidase kit (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA). Fluorescein isothiocyanate (FITC)–labeled goat anti-mouse IgG secondary antibody and propidium iodide were purchased from Chemicon; optimal cutting temperature (OCT) freezing compound (Tissue-Tek) from Sakura Finetek (Torrance, CA); and mounting medium (Vectashield) from Vector Laboratories.

All experimental procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Declaration of Helsinki for biomedical research involving human subjects and were approved by the Singapore National Eye Center and Singapore Eye Research Institute Ethics Committees.

Ultrathin PCL Film Preparation
Pellets of PCL were purchased from Sigma-Aldrich. This semicrystalline, biodegradable polymer had a melting index of 1.0 g/10 minutes, and a melting point of 60°C. The polymer pellets were kept in a desiccator before use.

The PCL pellets were dissolved in methylene chloride (6% wt/wt) and cast over glass sheets. The solvent was removed by slow evaporation for more than 6 hours. Membranes with initial thickness of 100 to 110 µm were obtained and were further vacuum dried at 51°C for 24 hours. Dried PCL membranes were pressed using a Cavar heat press (PH Hydraulics and Engineering, Singapore) with a platen temperature of 55°C, to remove surface defects. All pressed membranes were cut to 6 x 6 cm for biaxial drawing. Films were preheated for 30 minutes at 53 ± 1°C before drawing. Heated PCL membranes were biaxially stretched to 2.5 times the original size, at a speed of 200 mm/min, to obtain ultrathin PCL membranes 6 ± 2 µm in thickness.

Circular PCL membranes 14 mm in diameter were stamped out with a stainless-steel hollow punch (Elora, Rheinberg, Germany). One group of PCL membranes was treated with 5 M NaOH solution for 3 hours at room temperature to render the PCL membranes more hydrophilic. PCL membranes were sterilized by immersion in 70% ethanol and subsequently washed thrice with phosphate-buffered saline (PBS) and dried in an incubator at 37°C for 1 hour.

Atomic Force Microscopy and Scanning Electron Microscopy
Atomic force microscopy was performed with a system equipped with a dry scanner probe tip (TMX2000; Topometrix, Santa Clara, CA). All scans were performed over an area of 10 x 10 mm. Representative scans from three different membranes of each group were collected. For scanning electron microscopy, triplicates from each group were fixed in 2.5% glutaraldehyde for 4 hours at 4°C. They were then dehydrated in a graded ethanol series of 30%, 50%, 90%, and 100%, dried, and examined with a scanning electron microscope (JSM-580OLV; JEOL, Tokyo, Japan) at 15 W.

Tensile Testing
The tensile properties at room temperature were determined (model 4302 and 5542 tensile tester; Instron Universal, Norwood, MA). The sample size of each group was 10. Membranes were tested using a 5-N capacity load cell. The grip separation was set at 30 mm, and a testing speed of 5 mm/min was used. The biaxially drawn membranes were tensile tested at 45° to the biaxial stretch directions.

The thickness of the PCL membranes was measured with a spacer that had a known thickness (1 mm). The spacer provided large contact area over the soft membranes and prevented extraneous compression, commonly seen in conventional one-point contact measurements. The thickness of the film after biaxial stretching was measured with a mechanical comparator (MC 201-15; Sigma-Aldrich) to the nearest 0.5 µm. The engineering stress ({varsigma}) was defined as the ratio of load (L) to the sample cross-sectional area (A; {varsigma} = L/A). The percentage strain ({lambda}) was computed from [(l –l0)/L0] x 100%, where l was the total extension measured from the grip displacement and l0 the initial gauge length (40 mm). The initial Young’s modulus was calculated from the initial slope of the stress–strain curve. The tensile strength was obtained from the stress recorded at film fracture.

Water-Contact Angle Measurement
A telescopic goniometer (model 100-00(230); Rame-Hart, Mountain Lakes, NJ), was used to measure the static advancing water-contact angles of PCL membranes. Water-contact angles of untreated and NaOH-treated PCL membranes were measured at room temperature and 60% relative humidity, using a sessile drop (0.5 µL) method. More than five measurements were performed and the resulting values were averaged.

Preparation of HAM
Human placentas were obtained from mothers who had undergone cesarean sections. The membranes were washed with phosphate-buffered saline (PBS) to remove the blood clots. The HAM was peeled away from the chorion and flattened onto a sterilized nitrocellulose filter paper (Millipore, Bedford, MA). The HAM was then stored in 50% DMEM, 50% glycerol (Invitrogen-Gibco) at –80°C. In preparation for its use, the HAM was thawed, rinsed with PBS, and incubated with Dispase II (1.2 U/mL; Invitrogen-Gibco) for 2 hours. This was followed by gentle scraping to remove any remaining amniotic epithelial cells.

We evaluated the tensile strength and Young’s modulus of denuded HAMs. For our culture experiments, the HAMs were trimmed in size and placed epithelial basement membrane side up on culture dishes.

Cultivation of Rabbit Conjunctival Epithelial Cells
Conjunctival biopsy specimens were obtained from New Zealand Albino rabbits under anesthesia. Rabbit conjunctival epithelial cells were cultivated as cell suspension monolayers and as explants.

Cell Suspension Monolayer Cultures.
Rabbit conjunctival tissues were incubated in 1.2 U/mL Dispase II at 37°C for 2 hours. The epithelium was removed from the underlying stroma by gentle scraping and pipetting. After centrifugation at 1200 rpm for 10 minutes, the epithelial sheets were resuspended in 0.125% trypsin/0.02% EDTA and incubated for 10 minutes. Digestion was stopped with DMEM containing 10% FBS, followed by centrifugation. The cell pellets collected were resuspended in serum-free culture medium and plated onto untreated and NaOH-treated PCL membranes at a seeding density of 4 x 103 cells/cm2.

Explant Cultures.
Under an operating microscope, rabbit conjunctival epithelium was carefully dissected from the underlying stroma, cut into 0.5- to 1-mm pieces, and cultivated as explants on untreated and NaOH-treated PCL membranes in serum-free medium. The volume of medium was just sufficient to submerge the explants. When cellular outgrowth from the explants was observed, the volume of the medium was increased to fully immerse the explants.

The serum-free medium used consisted of keratinocyte growth medium supplemented with 10 ng/mL hEGF, 5 µg/mL insulin, 0.5 µg/mL hydrocortisone, 8.4 ng/mL cholera toxin, 30 µg/mL bovine pituitary extract, 50 µg/mL gentamicin, and 50 ng/mL amphotericin B. The cells were incubated at 37°C, under 5% CO2 and 95% air, with medium change performed every 2 days. The cells formed a confluent epithelial sheet on the PCL membranes after 8 to 12 days. The calcium concentration of the serum-free medium was subsequently increased to 1.2 mM with calcium chloride solution for a further 4 days to promote differentiation and stratification.

Morphology and Viability of Cultivated Cells
Cultures were monitored under an inverted phase-contrast microscope (Axiovert; Carl Zeiss Meditec, Inc., Oberkochen, Germany). The viability of cultivated cells was determined by staining with fluorescein diacetate (FDA; Molecular Probes, Inc., Eugene, OR). The cultures were incubated at 37°C with 2 µg/mL FDA in PBS for 15 minutes. After they were washed twice in PBS, each sample was then placed in 0.1 mg/mL propidium iodide solution for 2 minutes at room temperature to stain the nonviable cells. The samples were then washed twice in PBS and viewed under a confocal laser microscope (IX70-HLSH100 Fluoview; Olympus, Tokyo, Japan).

Proliferative Capacity of Cultivated Cells
The proliferative capacity of cultivated cells was evaluated by using the following methods.

BrdU ELISA Proliferation Assay.
Cells were cultured on untreated PCL membranes, NaOH-treated PCL membranes, and HAMs in 24-well plates with 500 µL culture medium per well. On day 6, cultured cells were incubated with 10 µM BrdU labeling solution for 20 hours at 37°C, followed by washing with 500 µL of PBS containing 10% serum per well. The cells were fixed with 70% ethanol in hydrochloric acid for 30 minutes at –20°C, and incubated with 300 µL of monoclonal antibody against BrdU for 30 minutes, followed by 300 µL of peroxidase substrate per well. The absorbance in each well was measured directly with a spectrophotometric microplate reader (spectrophotometer; Tecan, Grodig, Austria) at a test wavelength of 450 nm and a reference wavelength of 490 nm. Each sample was cultured in quadruplicate (n = 10).

Determination of Areas of Cellular Outgrowth from Explants.
The areas of cellular outgrowth from primary explants were measured on days 3, 5, 7, and 9, using computerized image measurement software (Axiovision KS300; Carl Zeiss Meditec, Inc.), until they reached the limit of the microscopic field (n = 8). We ensured that variances in outgrowth areas were not due to differences in the size of explants, because the mean areas occupied by the explants on untreated and NaOH-treated PCL membranes were similar (1.12 and 1.13 mm2, respectively).

Xenografting onto Severe Combined Immune-Deficient Mice
To evaluate the ability of cultured cells to continue to proliferate and stratify in vivo, the epithelial equivalents were xenografted onto the subcutaneous tissue of severe combined immune-deficient (SCID) mice. The SCID mice were anesthetized, and a dorsal skin flap was created to expose the underlying muscle fascia. Confluent cultures of epithelial cells on PCL membranes were placed epithelial-side up over the muscle fascia. The skin flap was returned to its original anatomic position and the wound edges sutured with 7-0 silk sutures. The mice were euthanized by asphyxiation with carbon dioxide 7 days after grafting, and the tissues excised for histologic analysis.

Histologic and Immunologic Analysis of Tissue Equivalents
Morphologic analysis of tissue equivalents was performed by first embedding them in OCT compound. Five-micometer sections were cut and stained with hematoxylin and eosin. Immunostaining was performed by incubating specimens with monoclonal antibodies to cytokeratins K4, K19, K3 (AE-5 antibody), MUC5AC, collagen IV, and integrin ß4 for 1 hour. MUC5AC was used to detect the gel-forming mucin present in conjunctival goblet cells. Normal mouse immunoglobulin and pancytokeratin (AE-1 and AE-3) were used as the negative and positive controls, respectively. The cells were subsequently incubated with secondary antibody (1:200 biotinylated horse anti-mouse immunoglobulin G) for 1 hour. These were detected by immunofluorescence by incubation with FITC-conjugated secondary antibody (goat anti-mouse IgG), followed by mounting (Vectashield; Vector Laboratories, Inc.). Immunostained cells were examined under a confocal laser microscope.

Goblet cell density was determined by counting the number of goblet cells as well as the total number of cells stained with propidium iodide in six representative high-power fields.21 We evaluated the goblet cell densities that were achieved for untreated and treated PCL membranes and compared it with normal in vivo rabbit conjunctiva as well as amniotic membrane cultures.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Surface Morphology of PCL Membranes
Biaxial stretching resulted in the formation of an ultrathin, almost transparent PCL membrane (Fig. 1A) . Atomic force microscopy demonstrated that, before stretching, the polymer molecules appeared as aggregates of spherulites (Fig. 1B) . The stretching process produced dense fibrils in the PCL membranes (Fig. 1C) . These fibrils were oriented mostly in a uniaxial direction and were packed closely together. Surface roughness increased from 190 to 349 nm after stretching.



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  		FIGURE 1. (A) Appearance of a biaxially stretched PCL membrane placed against a white background imprinted with the word "MEMBRANE", demonstrating its near transparency. Dotted line: the edge of the PCL membrane sheet. (B) AFM of an unstretched PCL membrane showing general unevenness of the membrane with a haphazard arrangement of polymer molecules appearing as aggregates of spherulites. (C) Atomic force microscopy appearance of a biaxially stretched PCL membrane, demonstrating the presence of closely packed, dense fibrils oriented mostly in a uniaxial direction (arrows).

 
Scanning electron microscopy showed that biaxial stretching resulted in an uneven surface topography and a fibrillary microstructure (Figs. 2A 2B) . PCL membranes that were surface modified with NaOH treatment showed areas of dimpling on the membrane surface (Fig. 2C) .



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  		FIGURE 2. SEM of PCL membranes. (A) The untreated PCL membrane possessed a diffusely uneven surface. (B) Biaxial stretching of the membrane resulted in the appearance of fibrils that were oriented in a uniaxial direction (arrows). (C) NaOH-treatment resulted in diffuse dimpling (arrows) over the surface of these membranes.

 
Tensile Testing and Water-Contact Angle Measurement
Before stretching, the tensile strength of PCL membranes was 21 ± 2 MPa, and the elongation at break was 676%. The tensile characteristics of the untreated and NaOH-treated PCL membranes are shown in Table 1 . After biaxial stretching, the tensile strength of PCL (untreated) membranes increased by almost twofold, to 42 ± 3 MPa, whereas the elongation at break decreased from 676% to 93%. These membranes were highly flexible and easy to handle.


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  		TABLE 1. Tensile Properties of Stretched PCL Membranes

 
NaOH treatment resulted in a slight decrease in tensile strength from 42 ± 3 to 35 ± 4 MPa. The relative Young’s modulus of the membranes before and after NaOH treatment was similar (225 ± 11 and 226 ± 11 MPa, respectively), whereas the elongation at break increased from 93% ± 9% to 166% ± 15% after treatment. NaOH treatment also resulted in a decrease in the water contact angle from 78° to 53°, indicating increased hydrophilicity of the membrane.

By comparison, HAMs had a tensile strength of 2.27± 0.01 MPa and a Young’s modulus of 1.61 ± 0.39 MPa. These were significantly lower than that of untreated and NaOH-treated PCL membranes.

Morphology and Viability of Cultivated Cells
Rabbit conjunctival epithelial cells began to migrate from the explants on the second day. The cells were small and round, with a prominent nucleus and scanty cytoplasm. The cells formed a densely populated epithelial sheet with an advancing border of loosely arranged cells. Cell suspension cultures formed colonies of small, ovoid cells. After 8 to 12 days, a confluent sheet of densely populated epithelial cells was formed. The cells demonstrated a healthy cobblestone morphology, with areas of stratification and differentiation. For both explant and cell-suspension culture methods, cells cultivated on NaOH-treated PCL membranes consisted of a more uniform sheet of round or ovoid cells, whereas those cultivated on untreated PCL membranes contained relatively more elongated cells (Figs. 3A 3B) . The morphology of cultivated cells on NaOH-treated PCL membranes was similar to that of amniotic membrane cultures (Fig. 3C) .



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  		FIGURE 3. Phase-contrast and SEM appearance of cultivated conjunctival epithelial cells on PCL membranes. (A) Cells cultivated on an untreated PCL membrane formed a confluent sheet of round and ovoid conjunctival epithelial cells, with areas that contained more elongated cells. (B) Cells cultivated on a NaOH-treated PCL membrane formed an epithelial sheet with a cobblestone morphology, consisting of fairly uniform round and ovoid cells. (C) Cells cultivated on amniotic membrane. The cell margins and cellular details were less distinct because of the less transparent nature of the amniotic membrane. SEM of cells cultivated on an untreated (D) and a NaOH-treated PCL membrane (E). Cells cultivated on NaOH-treated PCL membranes were found to be more uniform in size and were more densely confluent than were untreated membranes. (F) The epithelial sheet consisted of predominantly viable cells, as demonstrated by their positive staining for FDA (F). Bar (A–C) 100 µm; (D, E) 50 µm; (F) 100 µm.

 
SEM confirmed the presence of a confluent cell monolayer on the PCL membranes (Fig. 3D 3E) . Cells cultivated on NaOH-treated PCL membranes were more uniform in size and more densely confluent than were the untreated membranes, where more elongated cells were observed. Most of the cells in the epithelial sheet were confirmed to be viable, as demonstrated by their positive staining for FDA (Fig. 3F) .

Proliferation Assay
BrdU ELISA Proliferation Assay.
The proliferative capacity of conjunctival epithelial cells cultivated on untreated membranes was less than that of NaOH-treated PCL membranes (BrdU absorbance, 0.82 ± 0.07 and 1.05 ± 0.08, respectively; Fig. 4A) . This difference was statistically significant (t-test, P < 0.05). Conjunctival epithelial cells cultivated on HAM exhibited a greater proliferative capacity (BrdU absorbance, 1.36 ± 0.11) than did untreated and treated PCL cultures and these differences were also statistically significant (t-test, P < 0.05).



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  		FIGURE 4. Proliferation assays of cultivated conjunctival cells. (A) BrdU ELISA cell proliferation assay. The bars represent the BrdU absorbance for each substrate. There was greater cellular proliferation and incorporation of BrdU in cells cultivated on NaOH-treated membranes than in those on untreated membranes. Cellular proliferation was greatest in amniotic membrane cultures. (B) Areas of epithelial cell outgrowth from primary explants cultivated on untreated and NaOH-treated PCL membranes, demonstrating that NaOH-treated PCL membranes were more effective in supporting epithelial cell outgrowth and proliferation.

 
Areas of Outgrowth from Primary Explant Cultures.
The mean areas of cell outgrowth from explants cultivated on untreated and NaOH-treated PCL membranes are shown in Fig. 4B . The extent of cellular outgrowth from explants cultivated on untreated PCL membranes was correspondingly less than those cultivated on NaOH-treated PCL membranes (day 9 outgrowth areas, 666.7 ± 106.0 mm2 and 982.2 ± 34.7 mm2, respectively). This difference was statistically significant (t-test, P < 0.05).

Immunohistochemistry
The cultured conjunctival epithelial cells demonstrated a positive immunoreactivity for antibodies AE1 and AE3, and antibodies to K4 and K19 (Figs. 5A 5B 5C) . K3, a cornea-associated keratin, was not expressed by the conjunctival epithelial cells (Fig. 5D) . These findings were consistent with the normal conjunctival cytokeratin expression in vivo. Scattered cells that expressed the MUC5AC goblet cell mucin were observed on untreated PCL membranes and treated PCL membranes (Fig. 5E) . A large proportion of cells stained positively for Ki67, a marker for cell proliferation (Fig. 5F) . Collagen IV and ß4 integrin were expressed diffusely on the basement membrane side of the cultivated epithelial sheet (Figs. 5G 5H) .



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  		FIGURE 5. Immunostaining of cells cultivated on PCL membranes. The cultured conjunctival epithelial cells stained positively for (A) pancytokeratin (antibodies AE1 and AE3), (B) K4, and (C) K19. (D) K3 was not expressed by the conjunctival epithelial cells. (E) Scattered cells that expressed MUC5AC goblet cell mucin were observed (arrow). (F) Most of the cells stained positively for Ki67, a marker for cell proliferation. (G) Collagen IV deposition and (H) ß4 integrin were observed at the basal cell–substrate junction. Green: FITC staining of pancytokeratin, K4, K19, and MUC5AC. Red: propidium iodide staining of nuclei. Bar, 20 µm

 
Goblet Cell Density
Goblet cell density in normal in vivo rabbit conjunctiva was 21 ± 2.5 cells per 100 cells. The goblet cell densities for untreated and treated PCL membranes were 2.3 ± 0.8 and 3.0 ± 0.7 cells per 100 cells, respectively. This was comparable to the goblet cell density in amniotic membrane cultures (2.5 ± 1.1 cells per 100 cells). The differences between the goblet cell densities of the PCL and amniotic membrane substrate cultures were not statistically significant (P > 0.05).

Histologic Analysis of Conjunctival Epithelial Equivalents
Conjunctival epithelial cells formed a confluent epithelial sheet over untreated PCL membranes, consisting of one to two layers of flattened cells (Fig. 6A) . Cells cultivated on NaOH-treated membranes were more stratified and consisted of three to five layers of cells (Fig. 6B) . Xenotransplanted cultivated conjunctival-PCL membrane composite grafts underwent greater stratification and formed a multilayered epithelial sheet 8 to 10 cell layers in thickness, with cuboidal basal cells, and progressive flattening of the cells toward the surface (Fig. 6C) .



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  		FIGURE 6. Morphology of conjunctival epithelial equivalents. (A) Conjunctival epithelial cells cultivated on untreated membranes formed a confluent epithelial sheet consisting of one to two layers of flattened cells. (B) The epithelial sheet on NaOH-treated PCL membranes consisted of three to five layers of cells. (C) After transplantation into SCID mice, the epithelial sheet underwent greater proliferation and stratification and formed a multilayered sheet 8 to 10 cell layers in thickness, with cuboidal basal cells, and progressive flattening of the cells toward the surface. Arrows: PCL membranes. Bar, 200 µm.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Cultivated ocular surface equivalents have been used in the treatment of various ocular surface disorders, such as Stevens-Johnson syndrome and chemical injury.1 2 3 Ex vivo expansion techniques allow the formation of confluent sheets of cells from small tissue biopsy specimens, which is important for ocular conditions in which healthy tissue is deficient. Among the various biological membranes that have been used as substrates for cultivating ocular epithelial cells, HAM has been shown to demonstrate properties that are desirable for the treatment of ocular surface disease.1 2 3 4 5 However, several drawbacks related to the use of biological substrates include the inconsistency in tissue composition and the risks of transmission of infection and allograft rejection.

The ideal matrix material for the ocular surface should have the following characteristics: biocompatible, biodegradable, nontoxic, high tensile strength, ease of handling, able to support the growth of cells, pliable, and able to conform to various surfaces, and low in vitro and in vivo shrinkage. Using a bioresorbable ultrathin matrix material has distinct advantages. Thin membranes have higher permeability and therefore allow greater interaction between the graft and underlying tissue, allow more rapid diffusion of nutrients and substances, and reduce the amount of metabolic byproducts produced by degradation and resorption. In addition, thinner grafts are more flexible, can mold easily to the shape of various surfaces, and have greater transparency.22 23 These are particularly important properties for ocular surface transplantation. However, thin matrices are often associated with the problem of insufficient tensile strength and are often difficult to handle. Therefore, one of the challenges in ocular surface tissue engineering is to develop a biosynthetic membrane that is extremely thin, but at the same time has sufficient mechanical strength for transplantation purposes.

PCL, an aliphatic polyester that is bioresorbable and biocompatible, has been shown to be an ideal material for pharmaceutical products and wound dressings.17 18 19 20 The biaxial stretching technique resulted in a membrane that was ultrathin (<10 µm in thickness), which made the PCL highly flexible and conformable. At the same time the stretching process increased the tensile strength of the membrane by almost twofold. By comparison, HAM had a significantly lower tensile strength than that of stretched PCL membrane (42 and 2.27 MPa, respectively). The development of an ultrathin membrane that possesses sufficient mechanical strength to ensure easy handling represents a significant advancement in the field of bioengineering of ocular surface equivalents.17 18 19 20

The PCL membranes supported the attachment and proliferation of conjunctival epithelial cells, forming confluent stratified epithelial sheets. Although this was to a lesser degree than in the HAM cultures, the use of biosynthetic, biocompatible membranes offers several advantages in terms of eliminating the risk of disease transmission, reducing the inconsistency in tissue composition associated with biological substrates, being able to be custom fabricated to suit specific requirements, and possibly providing a readily available alternative tissue source for clinical use. The PCL membrane is also far more transparent than HAM. After transplantation, the cultivated cells underwent a greater degree of stratification and organization, suggesting that these cells remained highly proliferative in vivo. The ability for continued proliferation in vivo is extremely important for the long-term regeneration of the tissue after transplantation.

An added advantage of these biosynthetic membranes is the ability to perform surface modification to further enhance the biocompatibility of the material while keeping the bulk properties intact.24 25 Cultivated cells on untreated PCL membranes had a lower proliferative capacity compared with cells cultured on NaOH-treated membranes. This could be attributable to the fact that untreated PCL was more hydrophobic, as demonstrated by the higher water-contact angle of 78°. This hydrophobicity was likely to have affected the attachment and proliferative efficiency of these cells. Investigators have demonstrated that cultivated cells adhere and proliferate better at the more hydrophilic regions of biosynthetic membranes.26 27 28 Surface modification of the PCL membranes was performed with the use of NaOH. The hydrolysis converted the ester groups on the surface to more hydrophilic, hydroxyl, and carboxyl groups. NaOH treatment therefore rendered these membranes more hydrophilic and enhanced cell attachment and proliferation, as demonstrated by the greater proliferative capacity and improved morphologic appearance of cultivated cells.

A critical factor for bioengineered tissue equivalents is the ability of these tissue equivalents to retain their structural integrity after transplantation, which is dependent on basal cell attachment to the underlying substrate. The physical stretching process of the polymers resulted in the polymeric chains being stretched from their crystalline state into microfibrils. The fibrillary structure of the stretched membranes contributed to greater surface roughness, which was approximately two times greater than that of unstretched PCL. This higher surface roughness allowed a greater surface area for cell adhesion, which is important for enhancing cell-to-substrate attachment. In addition, these conjunctival epithelial equivalents on PCL membranes demonstrated the presence of collagen IV and ß4 integrin. Type IV collagen is a major component of basement membranes and hemidesmosomes contain the {alpha}6- and ß4-integrin complex. These properties are essential for maintaining graft integrity after transplantation.

Much of the literature on ocular surface tissue constructs involved culture methods that contain serum, with or without a 3T3 feeder cell layer.1 2 3 29 30 31 32 In the development of tissue equivalents for clinical transplantation, it is imperative that the use of animal serum and tissue be minimized, so as to reduce the possibility of contamination by infective agents and to avoid xenograft rejection. Under serum-free conditions, cultivated conjunctival cells formed confluent stratified epithelial sheets on the PCL membranes. Cultivation of epithelial cells in serum-free conditions without the use of animal feeder layers is a significant and important improvement over conventional methods of cultivating cells for transplantation.33 34 35 It provides a more defined condition with which to investigate the effect of various factors on the proliferation and differentiation of epithelial cells, reduces the risk of transmission of zoonotic infection, and minimizes the use of animal material in the culture process.

Conjunctival epithelial cells cultured on PCL membranes in serum-free medium were found to support goblet cell differentiation and proliferation, with NaOH-treated membranes having marginally greater goblet cell densities. The goblet cell densities achieved were comparable to that of amniotic membrane cultures. These results were consistent with our previous work on serum-free conjunctival cell cultures.21 34 35 It has been shown that goblet cell differentiation is likely to be tied to epithelial cell division and occurs at specific time points or after several cell divisions.32 Insufficient time in culture as well as the lack of specific growth-modulating factors in the culture system may have contributed to the lower goblet cell densities compared with the normal in vivo conjunctiva. In our study, goblet cells were observed to appear mainly after several days in culture. Their presence suggests that progenitor cells present in culture were able to retain their propensity toward goblet cell differentiation after several cell divisions. To date, no specific growth factors that selectively promote goblet cell differentiation have been identified. Selective propagation of goblet cells from primary cultures remains a subject for further investigation.

In summary, we demonstrated the use of a biaxially stretched ultrathin PCL membrane substrate for the development of a conjunctival epithelial equivalent. The membranes were highly flexible, easy to handle, and had a high strength-to-mass ratio. The PCL membrane supported the attachment and proliferation of conjunctival epithelial cells in culture as well as after transplantation. This promising material may have the potential to be used in tissue engineering ocular surface equivalents in the future. These findings have important clinical implications and are an important step toward the development of a safe and effective bioengineered tissue equivalent for clinical use.


   Acknowledgements
 
The authors thank Mary B. E. Chan-Park, School of Chemical and Biomolecular Engineering, and Jin Y. Shen, School of Mechanical and Aerospace Engineering, Nanyang Technological University, for their assistance in performing the mechanical stress studies.


   Footnotes
 
Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 2003.

Supported by NUS Grant R198/24/2000.

Submitted for publication April 26, 2005; revised September 7, 2005; accepted November 21, 2005.

Disclosure: L.P.K. Ang, None; Z.Y. Cheng, None; R.W. Beuerman, None; S.H. Teoh, None; X. Zhu, D.T.H. Tan, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Leonard P. K. Ang, Singapore National Eye Center, 11, Third Hospital Avenue, Singapore 168751; leopk{at}pacific.net.sg.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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