SCIENTIFIC ARTICLES J Oral Maxillofac Surg 62:980-988, 2004
Ex Vivo Produced Human Conjunctiva and Oral Mucosa Equivalents Grown in a Serum-Free Culture System Michiko Yoshizawa, DDS, PhD,* Stephen E. Feinberg, DDS, MS, PhD,† Cynthia L. Marcelo, PhD,‡ and Victor M. Elner, MD, PhD§ Purpose:
We sought to develop full-thickness ex vivo produced human conjunctiva and oral mucosa equivalents using a serum-free culture system without a feeder layer and to compare conjunctiva and oral mucosa equivalents to assess their suitability as graft materials for eyelid reconstruction. Materials and Methods: Human conjunctival and oral mucosal keratinocytes were cultured, expanded, and seeded onto AlloDerm (LifeCell Corp, Branchburg, NJ), a cadaveric, acellular dermis, to produce ex vivo produced full-thickness mucosa equivalents. Histology of equivalents and their expression of immunoreactive Ki-67, a proliferation marker, and GLUT1, a membrane antigen seen in barrier tissues, were examined at 4, 11, and 18 days after seeding onto AlloDerm. Results: Progressive epithelial stratification was observed on day 4, 11, and 18 conjunctiva and oral mucosa equivalents. Ki-67 immunoreactivity progressively increased with cultured time in both types of equivalent, indicating the continued presence of actively proliferating cells. GLUT1 immunoreactivity, concentrated in the basal keratinocytes of stratified epithelia of both types of equivalents, mimicked native tissue and indicated a high glycolytic state of the basal cells. Conclusions: Conjunctival and oral mucosal equivalents are similar to native tissue and demonstrate high proliferative and glycolytic states. Due to the similarity to conjunctiva, oral mucosal equivalents may be useful for eyelid reconstruction. Their advantages for surgical reconstruction include 1) ease of obtaining autogenous oral epithelium for expansion in vitro without the possibility of contaminating cellular- or serum-borne biologic agents, 2) growth of intact, confluent epithelia on rigid, transplantable human allogeneic dermis that may be surgically transplanted, and 3) reduced donor site morbidity and surgical time. © 2004 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 62:980-988, 2004 Eyelid reconstruction frequently requires augmentation of the conjunctiva as well as the cutaneous surfaces of the eyelids. Skin defects can be repaired by adjacent tissue transfers or with skin grafts, but conjunctiva mucosa, including the rigid tarsal conjunctiva, are frequently needed to reconstruct any defi-
ciencies of eyelid mucosal surface tissue. Conjunctiva mucosa may be replaced with autologous conjunctiva, oral mucosa, or nasal septal mucosa grafts. Conjunctiva surface expansion can be achieved with acellular spacers, including cadaveric sclera, autologous ear cartilage, and, recently, Medpor (Porex Surgical,
*Visiting Assistant Professor, Department of Oral and Maxillofacial Surgery, University of Michigan Medical Center, Ann Arbor, MI. †Professor, Department of Oral and Maxillofacial Surgery, University of Michigan Medical Center, Ann Arbor, MI. ‡Research Professor, Division of Plastic and Reconstructive Surgery, Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI. §Professor, Section of Oculoplastics, Department of Ophthalmology, University of Michigan Kellogg Eye Center, Ann Arbor, MI.
This study was supported in part by National Institutes of Health grant DE 13417 to Dr Feinberg. Address correspondence and reprint requests to Dr Feinberg: Department of Oral and Maxillofacial Surgery, University of Michigan Medical Center, B1-280TC, Box 0018, 1500 E Medical Center Dr, Ann Arbor, MI 48109-0018; e-mail:
[email protected] © 2004 American Association of Oral and Maxillofacial Surgeons
0278-2391/04/6208-0012$30.00/0 doi:10.1016/j.joms.2004.02.010
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Newnan, GA), AlloDerm (LifeCell Corp, Branchburg, NJ), or amniotic membrane. These grafts have a variety of disadvantages, including limited donor tissue availability, inadequate or excessive rigidity, donor site morbidity, and/or insufficient epithelialization resulting in persistently eroded mucosal surfaces. Presently, the material of choice is palatal mucosa; the major disadvantages are postoperative oral pain, donor site morbidity, and increased surgical and anesthesia costs.1-3 Although human and rabbit conjunctival epithelial cells have been successfully cultured with serum on collagen gels4,5 or 3T3 mouse fibroblast feeder cell layers,6,7 these epithelial cell layers cannot be used as grafts for reconstruction of the lower eyelid because they lack the rigidity necessary for transfer to surgical sites. In addition, the use of ex vivo produced grafts with transformed cell feeder layers and/or xenogeneic serum may introduce foreign agents into the ex vivo produced graft equivalent. A rigid, transplantable human mucosa graft produced ex vivo from autologous epithelial cells without a feeder layer or serum would be an ideal material for eyelid reconstruction. We previously reported the fabrication of ex vivo produced human oral mucosa equivalents using a serum-free culture system devoid of a feeder layer.8,9 In this study, we developed a full-thickness, ex vivo human conjunctival equivalent, composed of a confluent layer of conjunctival epithelial cells grown on a cadaveric acellular dermis, AlloDerm, in serum-free medium, without a feeder layer. We compared the histologic and immunoreactive characteristics of the conjunctival and oral mucosal equivalents with their native tissue to assess their suitability as autologous grafts.
Materials and Methods CELL CULTURE OF CONJUNCTIVAL KERATINOCYTES AND PRODUCTION OF CONJUNCTIVAL EQUIVALENTS
Conjunctiva tissue, obtained from patients undergoing ocular surgery, varied in size from 0.2 cm2 to 0.5 cm2. Tissue samples were transported in solution A (30 mmol/L hydroxyethylpiperazine-N⬘-2-ethanosulfonic acid, 10 mmol/L glucose, 3 mmol/L KCl, 130 mmol/L NaCl, 1.0 mmol/L Na2HPO4, pH 7.4),6 which was supplemented with 50 IU/mL penicillin, 50 g/mL streptomycin, and 2.5 g/mL amphotericin B (Fungizone; Sigma Chemical Co, St Louis, MO). Conjunctival tissue samples were digested with 0.04% trypsin (Sigma Chemical Co) solution, prepared in solution A, overnight at room temperature. The tissues were then separated above the basal layer, and the interface region was mechanically scraped gently
in an excess of a solution of 0.03% trypsin inhibitor (Sigma Chemical Co) to dissociate the basal cells from the submucosal layer. The resulting cell suspension was centrifuged and plated at 7.0 ⫻ 106 cells in 5 mL MCDB 153 medium per T-25 flask (Laboratory Science Co, Corning, NY) and incubated at 37°C in 5% CO2. MCDB 153 medium was prepared as described by Boyce and Ham10 and supplemented with 6.0 ⫻ 10⫺7 mol/L (0.218 g/mL) hydrocortisone, 5 ng/mL epidermal growth factor, 5 g/mL insulin (Sigma Chemical, Co), 6 mg/dL bovine pituitary extract (3 mL extract, 10 mg protein/mL) (Pel-Freez; Pel-Freez Biologicals, Rogers, AR), 25 g/mL gentamycin (Sigma Chemical Co), and 0.15 mmol/L CaCl2 to form “complete” growth medium.10,11 After the primary keratinocyte cultures were sufficiently expanded, they were seeded at cell densities of 1.25 ⫻ 105/cm2 on a piece of AlloDerm coated with type IV collagen (Life Technologies, Gaithersburg, MD). The equivalents were cultured submerged for 4 days and then raised to an air/liquid interface for 7 and 14 days using the organotypic tissue culture flasks (Organogenesis Inc, Canton, MA). These are referred to as day 4, day 11, and day 18 conjunctival equivalents. The culture medium used was MCDB 153 medium containing a high concentration of calcium, 1.8 mmol/L. CELL CULTURE OF ORAL KERATINOCYTES AND PRODUCTION OF ORAL EQUIVALENTS
We previously reported the use of primary oral keratinocytes and AlloDerm to create a culture system to produce oral mucosa equivalents.8,9 In brief, oral mucosa tissue samples obtained from patients undergoing dental extractions, preprosthetic surgeries, or gingivectomies were digested with 0.04% trypsin solution prepared in solution A, which was supplemented with 150 IU/mL penicillin, 150 g/mL streptomycin, and 7.5 g/mL amphotericin B, overnight, at room temperature. The tissues were then scraped to dissociate the basal cells from the submucosal layer in an excess of 0.03% solution of soybean trypsin inhibitor. Cells were cultured and seeded onto AlloDerm, and the resulting equivalents were cultured with the same technique as those for the production of conjunctival equivalents. These equivalents are referred to as day 4, day 11, and day 18 oral mucosal equivalents. HISTOLOGIC AND IMMUNOHISTOCHEMICAL STAINING
Samples of conjunctiva and oral mucosa tissues and conjunctiva and oral mucosa equivalents were fixed in 10% formalin, embedded in paraffin, cut at 5-m sections, and stained with hematoxylin and eosin. Selected sections were stained immunohistochemically for Ki-67 nuclear antigen (Ki-67) and HeptG2/
982 erythroid/brain–type glucose transporter (GLUT1). Ki-67 is a cell proliferation marker, and its expression is seen throughout the cell cycle except in G0.12 The sections were treated with 0.3% hydrogen peroxide in methanol and exposed to microwave pretreatment, which consisted of placing the sections in a pressure cooker filled with 0.01 mol/L citrate buffer (pH 6.0) and heating in a microwave for 14 minutes. After the sections were rinsed with 0.01 mol/L phosphate-buffered saline (PBS) (pH 7.2) supplemented with 0.5% skim milk and 0.05% Triton-X 100 (T-PBS) (Sigma Chemical Co), they were incubated with 5% skim milk in T-PBS for 1 hour to block nonspecific protein binding sites. Sections were incubated in primary antibodies at 4°C overnight. The following primary antibodies were used: monoclonal antibody to Ki-67 (MIB-1) (1:100) (Immunotech, Marseille, France) and polyclonal antibody to rabbit GLUT1 (1: 100) (Chemicon International, Temecula, CA). After the sections were rinsed with PBS, they were incubated in biotinylated horse anti-mouse IgG for Ki-67 and in biotinylated goat anti-rabbit IgG for GLUT1 at room temperature for 30 minutes. They were then rinsed and incubated in avidin-biotin peroxidase complex at room temperature for 30 minutes (Vector Laboratories, Burlingame, CA). After rinsing, they were treated with 0.02%, 3,3⬘-diaminobenzidine in 0.05 mol/L Tris-HCl buffer (pH 7.6) containing 0.05% hydrogen peroxide to visualize the reaction products. The specificity of the immunoreactions was checked by replacement of primary antibodies with nonimmune mouse IgG for Ki-67 and nonimmune rabbit IgG for GLUT1. The sections were counterstained with hematoxylin.
Results KERATINOCYTE CELL CULTURE OF CONJUNCTIVA AND ORAL MUCOSA
Native conjunctiva yielded about 5.5 ⫻ 106 cells/ cm2, and oral mucosa yielded about 7 ⫻ 106 cells/ cm2. Cells from both tissue types were grown and amplified in serum-free medium. Sufficient cells for primary seeding onto the AlloDerm were grown in vitro within 2 to 3 weeks. Morphologic features of the cultured conjunctival keratinocytes were polygonal cell profiles and small in size, similar to that previously reported for oral mucosal keratinocytes8,9 (Fig 1). HISTOLOGIC CHARACTERISTICS OF CONJUNCTIVAL NATIVE TISSUE AND IN VITRO EQUIVALENTS
Normal native conjunctival epithelium showed stratified squamous cells. Basal and midlayer cells were cuboidal, whereas more superficial cells as-
CONJUNCTIVA AND ORAL MUCOSA EQUIVALENTS
FIGURE 1. Phase-contrast photomicrograph of human conjunctival epithelial cells 4 days after plating. The cells form a mosaic pattern (scale bar, 25 m; original magnification ⫻400).
sumed flattened profiles. In the superficial layer, the flattened cells contained less conspicuous nuclei (Fig 2A). Conjunctival epithelium of day 4 equivalents was composed of 1- or 2-cell-thick continuous layers of flat cells on the AlloDerm (Fig 2B). The epithelial cells of day 11 equivalents, which were cultured submerged for 4 days and raised to air/liquid interface for 7 days, as previously described,8,9 had cell layers 1 to 3 cells thick (not shown). The squamous epithelium of day 18 equivalents, cultured submerged for 4 days and raised to air/liquid interface for 14 days,8,9 showed stratified cell layers up to 4 cells thick (Fig 2C). The superficial layer of the stratified epithelium showed inconspicuous nuclei, as seen in the native tissue. HISTOLOGIC CHARACTERISTICS OF ORAL MUCOSA NATIVE TISSUE AND IN VITRO EQUIVALENTS
Native oral mucosal epithelium consisted of stratified squamous cells. Basal cells were small and polygonal, increased in size in the midlayers of the epithelium, and showed progressive flattening in the superficial layers. The superficial cells were flat, exhibited increased eosinophilia due to keratinization, and contained smaller and less conspicuous nuclei (Fig 3A). As previously reported,8,9 the epithelium of day 4 equivalents showed a continuous monolayer of keratinocytes (Fig 3B). At day 11, the epithelium on the equivalent was 3 to 6 cells thick (not shown). By day 18, the squamous epithelium was stratified up to 8 cells thick and showed superficial keratinization (Fig 3C). IMMUNOHISTOCHEMICAL FINDINGS OF CONJUNCTIVAL NATIVE TISSUE AND IN VITRO EQUIVALENTS
Immunoreactivity for Ki-67 nuclear antigen was present in all layers of native conjunctival epithelium
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in flat endothelial cells of substantia propria blood vessels. Virtually all of the epithelial cells of day 4, 11, and 18 conjunctival equivalents were immunopositive (Figs 5B, C). IMMUNOHISTOCHEMICAL FINDINGS OF ORAL MUCOSA NATIVE TISSUE AND IN VITRO EQUIVALENTS
Immunoreactivity for Ki-67 nuclear antigen was seen in the basal and suprabasal layers of native oral
FIGURE 2. Native human conjunctiva mucosa and ex vivo equivalents (hematoxylin and eosin stain; scale bar, 25 m; original magnification ⫻400). A, Native epithelium consists of nonkeratinized stratified squamous cells. B, Day 4 equivalent consists of a continuous epithelial cell layer on AlloDerm. C, Day 18 equivalent consists of a stratified epithelial layer of flattened keratinocytes, 2 to 4 cells thick on AlloDerm.
(Fig 4A). Focal epithelial cells of day 4 equivalents showed nuclear immunoreactivity (Fig 4B). Progressively increased numbers of immunoreactive epithelial nuclei were evident in day 11 and 18 equivalents (Fig 4C). The majority of native conjunctival epithelial cells were immunopositive for GLUT1 (Fig 5A). Immunoreactivity was more intense in many of the basal and suprabasal cells. Immunopositivity was also seen
FIGURE 3. Native human oral mucosa and ex vivo equivalents (hematoxylin and eosin stain; scale bar, 25 m; original magnification ⫻400). A, Native epithelium consists of a multilayer of stratified squamous cells. B, Day 4 equivalent consists of a continuous monolayer of cells on AlloDerm. C, Day 18 equivalent consists of a stratified epithelial layer, up to 8 cells thick, with keratinization on AlloDerm.
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GLUT1 (Fig 7A). Immunoreactivity was limited to the deeper layers of the epithelium and was lacking in the superficial keratinized layer (Fig 7B). All epithelial cells of days 4 (Fig 7C) and 11 (not shown) equivalents showed immunopositive GLUT1 reactivity. Day
FIGURE 4. Ki-67 immunoreactivity of native conjunctival mucosa and ex vivo equivalents (counterstained with hematoxylin; scale bar, 25 m; original magnification ⫻400). A, Native epithelium exhibits immunopositive cells in basal, suprabasal, and superficial layers. B, Day 4 equivalent shows focal immunopositivity in basal layer (arrowhead). C, Day 18 equivalent exhibits several immunopositive cells (arrowheads).
mucosal epithelium (Figs 6A, B). Focal nuclei of cells in the basal layer of day 4 equivalents were immunopositive (Fig 6C). Day 11 and 18 oral mucosa equivalents showed progressive increases in the number of immunopositive nuclei in the deeper layers of the epithelia (Fig 6D). Native oral mucosa epithelium exhibited strong immunohistochemical staining for
FIGURE 5. GLUT1 immunoreactivity of native conjunctival mucosa and ex vivo equivalents (counterstained with hematoxylin; scale bar, 25 m; original magnification ⫻400). A, Native epithelium exhibits immunopositivity in all layers, with intense expression in basal and suprabasal layers (arrow). Immunopositivity was also seen in flat endothelial cells of substantia propria blood vessels (arrowheads). B, Day 4 equivalent shows diffuse immunopositivity. C, Day 18 equivalent exhibits diffuse immunopositivity with a more intense staining in basal layer (arrow).
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FIGURE 6. Ki-67 immunoreactivity of native oral mucosa and ex vivo equivalents. A, B, Native epithelium exhibits immunopositive cells in basal and suprabasal layers. A–D counterstained with hematoxylin (scale bar: A, 10 m, original magnification ⫻100; B, 25 m, original magnification ⫻400). C, Day 4 equivalent shows focal basal immunopositive reaction (arrow) (scale bar, 25 m, original magnification ⫻400). D, Day 18 equivalent exhibits numerous immunopositive cells in basal and suprabasal layers (scale bar, 25 m, original magnification ⫻400).
18 equivalents mimicked native oral mucosa with strong basilar and mid-layer immunoreactivity with absence of immunoreactivity in superficial, keratinized epithelial cells (Fig 7D).
Discussion A tarsal-conjunctival graft is an ideal composite graft for lower eyelid reconstruction. However, adequate tissue often cannot be obtained without damaging the host lid.1 Hard palatal mucosa has been found to be useful for lower eyelid reconstruction because it contains dense collagen in the submucosal lamina propria, which imparts rigidity.1-3 The major disadvantages of hard palatal grafting are added intraoperative complexity, postoperative discomfort, and a tissue defect that must heal via secondary intention. To our knowledge, this is the first study to describe ex vivo produced conjunctival equivalents comprised of human conjunctiva epithelium grown on a transplantable cadaver dermis, AlloDerm, without a feeder layer or the use of serum. Previously, all conjunctival
epithelial cell cultures have required the use of serum, fibroblasts, or 3T3 cells and have never been grown on rigid dermal supports.4-7,13-16 Reported culture methods include human and rabbit conjunctival epithelial cell cultures grown using a 3T3 feeder cell layer6,7,16 or rabbit and human cell cultures using collagen gels,4,5 with the latter containing either human conjunctival fibroblasts, 3T3 cells, or no cells.4 The only disadvantage of our present culturing protocol is the inclusion of pituitary extract in our culture medium. Pituitary extract could be a potential source for contamination of our autologous keratinocytes with prions or slow virus. We have recently been successful in eliminating this undefined supplement in our present protocol for the ex vivo assembly of our tissue equivalents. The epithelium of conjunctival equivalents gradually stratified with time, but the number of cell layers was fewer than that seen in oral mucosal equivalents, consistent with differences we observed in the native tissues from which the primary cultures were derived. At day 18, epithelium of both types of equiva-
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FIGURE 7. GLUT1 immunoreactivity of native oral mucosa and ex vivo equivalents. A, B, Native epithelium exhibits an immunopositivity in all except superficial layer, with most intense reactivity along basal layer. A–D counterstained with hematoxylin (scale bar: A, 10 m, original magnification ⫻100; B, 25 m, original magnification ⫻400). C, Day 4 equivalent shows diffuse immunopositivity (scale bar, 25 m, original magnification ⫻400). D, Day 18 equivalent exhibits intense immunopositivity in basal and suprabasal layers but lacks expression in superficial keratinocytes (scale bar, 25 m, original magnification ⫻400).
lents had stratified, with the surface layers consisting of flattened cells with keratinization. The oral mucosal equivalents showed more abundant cuboidal cells in the basal layers. Ki-67–positive cells were present principally in the basal and suprabasal layers of the native conjunctiva epithelium and conjunctiva equivalents as well as in native gingiva and oral mucosal equivalents, as we previously reported.9 Of note, Ki-67 immunoreactivity was higher in both types of equivalents than was seen in their native tissue. This may relate to the fact that Ki-67 is a nuclear DNA-associated antigen expressed in G1, S, G2, and M phases of proliferating cells but absent in G0 resting cells.17,18 Accordingly, Ki-67 immunopositivity has been previously reported mainly in the basal and suprabasal cell layers of native conjunctival and gingival epithelium.9,19,20 A hyperproliferative state of oral keratinocytes within oral mucosa equivalents was postulated by Chung et al,18 who found that basal cells of epithelium grown on deepidermalized dermis were more immunoreactive for proliferating cell nuclear antigen than were native oral buccal mucosal basal cells. Our
data showing enhanced Ki-67 immunoreactivity within the basal cells of both oral and conjunctival equivalents also suggest a more hyperproliferative state than found in the respective native tissues. GLUT1 is one isoform of 6 mammalian facilitative glucose transporters that mediate the transport of glucose down its chemical gradient across the plasma membrane.21-23 GLUT1 is expressed in several cell barriers, including the blood-brain and blood-eye barriers.24-29 GLUT1 is also present along the basilar layers of human epidermis, oral mucosa, and conjunctiva, where its expression seems to be related to barrier functions and keratinocyte differentiation.7,30 We found GLUT1 immunopositivity was most intense in the basilar layers of native conjunctival and oral mucosal epithelium, with the latter devoid of immunoreactivity in the superficial keratinized cells. GLUT1 positivity was present in all cells of the conjunctiva equivalents at 4, 11, and 18 days. In contrast, oral mucosa equivalents showed stratification with loss of superficial GLUT1 positivity at day 18, mimicking native tissue. Gherzi et al,7,30 also reported high GLUT1 expression in the basal layers of human con-
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junctiva, oral mucosa, and skin epithelia reconstituted in cultures containing 3T3-J2 cells and fetal calf serum. They found that GLUT1 expression was 4- to 6-fold higher in the basal cells compared with the superficial, differentiated keratinocytes. They also found that culture conditions promoting keratinoctye differentiation reduced GLUT1 immunoreactivity. Based on these findings, they postulated that GLUT1, located along the basal epithelial barrier, might play an important role in mediating nutrient and fluid supply to the upper layers of the epithelium from the underlying vascularized stroma.7 The intense, diffuse GLUT1 immunopositivity we found in the conjunctiva and oral mucosal equivalents at days 4 and 11 and persistent, diffuse positivity in conjunctiva equivalents at day 18 suggest high glucose metabolism in these epithelial equivalents. Voldstedlund and Dablesteen31 postulated that GLUT1 expression is due to high glucose-dependent metabolism in stratified squamous epithelia, particularly at the edges of mucosal wounds where proliferation is increased. In contrast, they found that mucosal epithelium normally exhibits intense GLUT1 reactivity only in the basal third, with weak superficial layer positivity, similar to our day 18 oral mucosa equivalents.31 The reduced GLUT1 expression represents a shift from glucose-dominated to fat-dominated metabolism during the differentiation process of basal cells into superficial keratinocytes.32 The superficial keratinocytes of native oral mucosa and oral mucosal day 18 equivalents exhibited findings similar to that described by Voldstedlund and Dablesteen,31 reduced GLUT1 expression, which indicates that our equivalents mimic the differentiation found in native tissue. The photomicrographs presented here show that the native mucosal tissue is more stratified than either the tissue-engineered conjunctiva or oral mucosal equivalents. It is known that the stratified epithelium of the mucosa plays an important role as a barrier tissue. As a barrier, the epithelial layer prevents microbial and other foreign materials from entering into the body as well as formulating a pathway for diffusion of nutrients and cytokines.33 The barrier effect from an intact epithelial layer was evident in our preliminary human clinical trials.34 In this study, there was a marked intradermal inflammatory response in the patient cohort, which did not receive a dermal equivalent with an epithelial layer, compared with the experimental group, which received a graft with the oral mucosa equivalent containing an intact epithelial layer.34 As we know from our clinical experience with free oral mucosal grafts, the more differentiated superficial stratified layers of epithelium will degenerate or “slough off” after grafting, leaving only the basal cells, because they have no ability to divide. Our studies in SCID mice35 have shown that once trans-
planted as a free graft, the “thin” epithelial layer, composed of mostly basal cells, which are either stem or transit amplifying cells, continue to divide and stratify in vivo. Therefore, the initial presence on a grafted mucosal tissue-engineered equivalent of a thin epithelial cell layer should not be an impediment to clinical maturation of the epithelial layer after transplantation to the recipient site. In fact, a thicker epithelial layer will not be beneficial, because it will take longer to fabricate the construct and the additional layers will eventually be lost after grafting, thus offering no advantage. Our results indicate that day 18 conjunctival and oral mucosal equivalents, grown ex vivo, without serum or a feeder layer, are similar to their respective native tissue. The epithelia of the equivalents show high proliferative and glycolytic states as indicated by the presence of both Ki-67 nuclear antigen and GLUT1 immunoreactivity, particularly within proliferating basal keratinoctyes. Due to their similarity to native conjunctiva, oral mucosal equivalents may be useful for eyelid reconstruction. The advantages of oral mucosal equivalents for surgical reconstruction include 1) the ease of obtaining autogenous oral mucosal epithelium for keratinocyte amplification in an environment without the possibility of contaminating cellular- or serum-borne biologic agents, 2) growth of intact, confluent epithelia on a rigid, transplantable human allogeneic dermis that can be surgically transplanted, and 3) reduced donor site morbidity and surgical time. Acknowledgments The authors would like to thank Lenore Rhodes, Kathy McClinchey, and Nancy Smith for their excellent technical assistance.
References 1. Kohen MS, Shorr N: Eyelid reconstruction with hard palate mucosa graft. Ophthalmic Plast Reconstr Surg 8:183, 1992 2. Siegel RJ: Palatal grafts for eyelid reconstruction. Plast Reconst Surg 76:411, 1985 3. Hatoko M, Kuwahara M, Shiba A, et al: Reconstruction of full-thickness lower eyelid defects using a blepharoplasty technique with a hard palate mucosal graft. Ann Plast Surg 42:688, 1989 4. Tsai RJF, Ho YS, Chen JK: The effects of fibroblasts on the growth and differentiation of human bulbar conjunctival epithelial cells in an in vitro conjunctival equivalent. Invest Ophthalmol Vis Sci 35:2865, 1994 5. Niiya A, Matsumoto Y, Ishibashi T, et al: Collagen gel embedding culture of conjunctival epithelial cells. Graefe’s Arch Clin Exp Ophthalmol 235:32, 1997 6. Wei ZG, Lin T, Sun TT, et al: Clonal analysis of the in vivo differentiation potential of keratinocytes. Invest Ophthalmol Vis Sci 38:753, 1997 7. Gherzi R, Melioli G, Luca MD, et al: High expression levels of “erythroid/brain” type glucose transporter (GLUT1) in the basal cells of human eye conjunctiva and oral mucosa reconstituted in culture. Exp Cell Res 195:230, 1991 8. Izumi K, Takacs G, Terashi H, et al: Ex vivo development of a composite human oral mucosal equivalent. J Oral Maxillofac Surg 57:571, 1999
988 9. Izumi K, Terashi H, Marcelo CL, et al: Characterization of an ex vivo produced human oral mucosal composite developed in a serum-less culture system. J Dent Res 79:798, 2000 10. Boyce ST, Ham RG: Cultivation, frozen storage, and clonal growth of normal epidemal keratinocytes in serum-free medium. J Tissue Culture Methods 9:83, 1985 11. Boyce ST, Ham RG: Normal human epidermal keratinocytes, in Weber MM, Sekely L (eds): In Vitro Models for Cancer Research. Boca Raton, FL, CRC Press, 1985, p 245 12. Sasaki K, Murakami T, Kawasaki M, et al: The cell cycle associated change of the Ki-67 reactive nuclear antigen expression. J Cell Physiol 133:579, 1987 13. Diebold Y, Calonge M, Ferna´ndez N, et al: Characterization of epithelial primary cultures from human conjunctiva. Graefe’s Arch Clin Exp Ophthalmol 235:268, 1997 14. Saha P, Uchiyama T, Kim KJ, et al: Permeability characteristics of primary cultured rabbit conjunctival epithelial cells to low molecular weight drugs. Curr Eye Res 15:1170, 1996 15. Saha P, Kim KJ, Lee VHL: A primary culture model of rabbit conjunctival epithelial cells exhibiting tight barrier properties. Curr Eye Res 15:1163, 1996 16. Tsai RJF, Tseng SCG: Substrate modulation of cultured rabbit conjunctival epithelial cell differentiation and morphology. Invest Ophthalmol Vis Sci 29:1565, 1988 17. Gerdes J, Lemke H, Baisch H, et al: Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710, 1984 18. Chung JH, Cho KH, Lee DY, et al: Human oral buccal mucosa reconstructed on dermal substrates: A model for oral epithelial differentiation. Arch Dermatol Res 189:677, 1997 19. Bernauer W, Wright P, Dart JK, et al: Cytokines in the conjunctiva of acute and chronic mucous membrane pemphigoid: An immunohistochemical analysis. Graefe’s Arch Clin Exp Ophthalmol 231:563, 1993 20. Nishida K, Yamanishi K, Yamada K, et al: Epithelial hyperproliferation and transglutaminase 1 gene expression in StevenJohnson syndrome conjunctiva. Am J Pathol 154:331, 1999 21. Bell GI, Kayano T, Buse JB, et al: Molecular biology of mammalian glucose transporters. Diabetes Care 13:198, 1990 22. Mueckler M: Facilitative glucose transporters. Eur J Biochem 219:713, 1994 23. Bell GI, Burant CF, Takada J, et al: Structure and function of mammalian facilitative sugar transporters. J Biol Chem 268: 19161, 1993
CONJUNCTIVA AND ORAL MUCOSA EQUIVALENTS 24. Pardridge WM, Boado RJ, Farrell CR: Brain-type glucose transporter (GLUT1) is selectively localized to the blood-brain barrier. Studies with quantitative Western blotting and in situ hybridization. J Biol Chem 265:18035, 1990 25. Harik SI, Kalaria RN, Whitney PM, et al: Glucose transporters are abundant in cells with “occluding” junctions at the bloodeye barriers. Proc Natl Acad Sci U S A 87:4261, 1990 26. Takata K, Kasahara T, Kasahara M, et al: Erythrocytes/HepG2type glucose transporter is concentrated in cells of blood-tissue barriers. Biochem Biophys Res Commun 173:67, 1990 27. Takata K, Kasahara T, Kasahara M, et al: Ultracytochemical localization of the erythrocyte/HepG2-type glucose transporter (GLUT1) in the ciliary body and iris of the rat eye. Invest Ophthalmol Vis Sci 32:1659, 1991 28. Takata K, Kasahara T: Ultracytochemical localization of the erythrocyte/HepG2 type glucose transporter (GLUT1) in cells of the blood-retinal barrier in the rat. Invest Ophthalmol Vis Sci 33:377, 1992 29. Kumagai AK, Glasgow BJ, Paradridge WM: GLUT1 glucose transporter expression in the diabetic and nondiabetic human eye. Invest Ophthalmol Vis Sci 35:2887, 1994 30. Gherzi R, Melioli G, Luca MD: “HepG2/erythroid/brain” type glucose transporter (GLUT1) is highly expressed in human epidermis: Keratinocyte differentiation affects GLUT1 levels in reconstituted epidermis. J Cell Physiol 150:463, 1992 31. Voldstedlund M, Dablesteen E: Expression of GLUT1 in stratified squamous epithelia and oral carcinoma from human and rats. Acta Pathol Microbiol Immunol Scand 105:537, 1997 32. Freinkel RK: Carbohydrate metabolism of epidermis, in Lowell A, Goldsmith MD (eds): Biochemistry and Physiology of the Skin (ed 2). New York, NY, Oxford University Press, 1991, pp 452-461 33. Falanga V, Margolis D, Alvarez O, et al: Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Arch Dermatol 134:293, 1998 34. Izumi K, Feinberg SE, Iida M, et al: Intraoral grafting of an ex vivo produced oral mucosa equivalent: A preliminary report. Int J Oral Maxillofac Surg 32:188, 2003 35. Izumi K, Feinberg SE, Terashi H, et al: Evaluation of transplanted tissue-engineered oral mucosa equivalents in severe combined immunodeficient mice. Tiss Eng 9:163, 2003
