Skin and oral mucosal substitutes


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Skin and oral mucosal substitutes

  1. 1. Skin and oral mucosal substitutesKenji Izumi, DDS, PhDa, Stephen E. Feinberg, DDS, MS, PhDb,*aDepartment of Tissue Regeneration and Reconstruction, Division of Reconstructive Surgery for Oral and Maxillofacial Region,Niigata University Graduate School for Medical and Dental Sciences, Niigata City, Niigata, JapanbDepartment of Oral and Maxillofacial Surgery, University of Michigan Medical Center,B1–A 235 UH, Box 0018, 1500 East Medical Center Drive, Ann Arbor, MI 48109, USABiologic substitutes of human skin and mucosahave several prospective applications, including, butnot limited to, (1) models of skin and mucosal biol-ogy and pathology, (2) treatment and closure of skinand mucosal wounds, (3) alternatives to animals forsafety testing of consumer products, and (4) deliveryand expression of transfected genes [3]. This articleintroduces the reader to the types of skin and mucosasubstitutes that have been and are being developed inthe area of tissue engineering for use in proceduresfor trauma, ablative oncologic resections, and recon-structive surgery.Skin and mucosal substitutes have a common setof requirements for the duplication of anatomicstructures and physiologic functions that they are toemulate. For use in wound closure, the first definitiverequirement is re-establishment of the epidermalbarrier to fluid loss and microorganisms and alle-viation of pain and enhancement of wound healing.In full-thickness skin loss, replacement of the epi-dermis and dermis is the preferred approach. Replace-ment of these tissue components also must minimizescar formation and restore acceptable function andcosmesis. A major advantage of the use of substitutesin wound closure is reduction or elimination of thedonor site for skin and mucosal grafts. Success in theelimination or minimization of donor site morbiditycould shorten recovery time and reduce the length ofoperative procedures.Approaches that have been used in the fabrication,manufacturing, and ‘‘tissue engineering’’ of skin andmucosa substitutes can be classified as (1) in vitroculturing of autologous and allogeneic keratinocytes,(2) in vitro tissue engineering of dermis composed ofeither artificial (collagen, glycosaminoglycans, poly-mers of polyglycolic, and polylactic acid) or alloge-neic and acellular dermis, and (3) a bilayer of skinmucosa from a combination of (1) and (2). Threeessential components are known to be necessary toengineer human skin and mucosa: cells, an extra-cellular matrix, and cytokines [30,42].Skin substitutesA major milestone in the development of skinsubstitutes was the introduction of the in vitro tech-nique of Rheinwald and Green [47] that involved theculturing of human keratinocytes into epithelialsheets suitable for autografts. These investigatorsused a combination of hydrocortisone, epidermalgrowth factor, and irradiated murine 3T3 fibroblaststo support the proliferation of keratinocytes on plasticsubstrates. The following ingredients were addedto improve the culture media and to facilitate kerati-noctye sheet formation: (1) insulin, to promote theuptake of glucose and amino acids; (2) transferrin, todetoxify iron; (3) hydrocortisone, to promote theattachment of cells and cell proliferation; (4) triiodo-thyronine, as a mitogen for keratinocytes; and (5)cholera toxin, to upregulate cAMP levels. This1042-3699/02/$ – see front matter D 2002, Elsevier Science (USA). All rights reserved.PII: S1042-3699(02)00010-9This work was supported by a Grant-in-Aid forScientific Research (No. 12771216) from the Ministry ofEducation, Science and Culture, Japan (KI) and from GrantNo. DE13417 from the National Institute of Health,USA (SEF).* Corresponding author.E-mail address: (S.E. Feinberg).Oral Maxillofacial Surg Clin N Am 14 (2002) 61–71
  2. 2. method allowed keratinocytes to grow into a largeepithelium sheet — 10,000 times larger than the ori-ginal biopsy — in a short time period, enough tocover the large body surface areas. Investigators werethen able to release sheets of keratinocytes by usingthe enzyme dispase, which could digest adhesivemolecules holding the keratinocytes to the plasticsubstrate without digesting the cohesive links be-tween the keratinocytes themselves. O’Connor et al.[39] reported the first human use of autologouscultured keratinocyte sheet grafts for burn wounds.Cultured keratinocyte sheets have been applied invarious clinical scenarios such as chronic skin ulcers[51], congenital nevi [20], and junctional epider-molysis bullosa [5]. This approach revolutionizedsurgical treatment of burns and was the forerunnerto the impetus to engineer skin to enhance tissueregeneration and repair.Two major approaches are currently used for invivo tissue engineering of skin and mucosa. The oldertechnique of Rheinwald and Green [47] is based onthe use of a serum-containing medium and a feederlayer of lethally irradiated transformed cell line ofmouse fibroblasts. The second approach relies onserum-free media in the absence of a feeder layer.The presence of irradiated feeder cells and serum canbe a confounding factor in keratinoctye cultures whenused for experimental research. This technique alsowould have difficulties receiving Food and DrugAdministration (FDA) approval because of the poten-tial transmission of unknown elements such as slowviruses to graft recipients. For this reason, manyinvestigators have tried to avoid using feeder cellsand to reduce the amounts of serum and additives,such as pituitary extract.The various methods for in vitro engineering ofthe epidermis require the use of (1) coating ofculture surfaces with molecules found in the extra-cellular matrix, such as collagen, fibronectin, or lam-inin, that assist in simulating in vivo conditions and(2) variable concentrations of calcium in the culturemedium. Calcium ions play a vital role in thegrowth and differentiation of keratinocytes. Increas-ing calcium concentration is accompanied by anincreasing level of keratinocyte differentiation, asevidenced by increasing numbers of formed desmo-somes and the formation of multilayers and sheets ofcells. In vitro engineering of the epidermis alsorequires (3) the use of complex biologic extractssuch as bovine pituitary extract and (4) the additionof mitogens or trace elements.For a skin or mucosa substitute to obtain FDAapproval, it is necessary to eliminate the use of serumand xenogeneic feeder layers, undefined biologicextracts such as pituitary extract, and products thatmay be modes of transmission of diseases, such as anybovine products that are not certified from disease-free herds. Several skin substitutes are approved bythe FDA and are commercially available.EpitheliumEpicel (Genzyme Tissue Repair Corp, Cam-bridge, MA) is composed of cultured epithelialautogenous keratinocyte sheets and has been com-mercially available since 1988. The procedure ofgenerating cultured epithelial sheets follows the tech-nique of Rheinwald and Green [47]. The epithelialsheets are composed of several stratified layers ofkeratinocytes that are formed by culturing submergedcells (totally covered by medium) for 10 to 15 days.An epithelial sheet produced by their culture systemhas several advantages. The first advantage is thepossibility of a large expansion from a small donorsite of 2 cm2up to 10,000-fold, which could covera full-surface body area of an adult, approximately2 m2. The second advantage is the low risk oftransmission of viral diseases such as bovine spongi-form encephalopathy. Early clinical studies of epi-thelial sheets used for the treatment of burn woundshad encouraging outcomes [19,37]. Later clinicalexperiences, in contrast, demonstrated low graft‘‘take’’ rate [16,48]. Disadvantages of cultured sheetsof grafted keratinocytes include (1) time-consuminggrowth of keratinocyte sheets in which, even underoptimal conditions, it would take 2 to 3 weeks toobtain a sufficient amount of keratinocyte sheets forgrafting; (2) the use of potentially immunogenicmaterials in culture, such as serum, various additives(pituitary extract), and a xenogeneic feeder layer thatmay contribute to graft loss; (3) widely reportedvariations in the ‘‘take’’ of keratinocyte sheet grafts,which is lower than that of split-thickness grafts; (4)graft instability, such as graft fragility and blisterformation, that may be secondary to the absence ofrete ridges and inadequate formation of epidermalkeratinocytes’ anchoring fibrils; (5) wound contrac-tion, which is secondary to a difference in specificcharacteristics of the keratinocytes within the cul-tured sheets to that of normal in situ keratinocytesand to the lack of dermal component within culturedepithelial sheet graft [10,44]; and (6) the cost of thegrafts production.DermisDermis plays several biologic and functionalroles in the skin. The most important roles are (1)K. Izumi, S.E. Feinberg / Oral Maxillofacial Surg Clin N Am 14 (2002) 61–7162
  3. 3. providing mechanical support for cells involved inskin structure formation, immunity, nutrition, andsensation; (2) providing skin elasticity and tensilestrength; and (3) functioning as an anchor forepithelial glands and keratinizing appendage struc-tures of the skin (hair, nails). During skin healing,the presence of dermis also supports faster reepithe-lialization, inhibits wound contraction, and improvesesthetic outcome. Increased understanding of dermalstructure and composition has guided the develop-ment of artificial dermal substitutes. Structure isonly one of the material property requirements.Dermis also should be pliable, hemocompatible,minimally immunogenic, and eventually degradableand must minimize fluid loss and reduce scarringand contractures.In designing a dermal replacement, one must takeseveral clinical observations into consideration: (1)the thicker the dermal layer of a split-thickness skingraft, the less the graft contracts; (2) full-thicknessskin grafts contract minimally; (3) full-thicknessdermal injuries heal by contraction and hypertrophicscarring, which produce subepithelial scar tissue thatis nothing like the original dermis; (4) partial-thick-ness wounds with superficial dermal loss heal withless hypertropic scarring; and (5) the length of illnessin burn cases is essentially restricted to the length oftime the burn wound is open. One might hypothesizefrom these observations that the dermis providesinformation to the wound that modulates the healingprocess. If so, then a dermal replacement shouldprovide the information necessary to control theinflammatory and contractile processes and the in-formation necessary to evoke ordered re-creation ofautologous tissue in the form of a neodermis. Theinitial replacement material also should provideimmediate physiologic wound closure and be elimi-nated once it has provided sufficient information forreconstitution of a neodermis.Yannas, Burke, and colleagues [4,22,55] focusedon these observations and developed a bilayered two-stage model that resulted in a FDA-approved productthat is marketed as Integra (Integra Life SciencesCorp, Plainsboro, NJ). Integra has a top layer of asilicone elastomer and a bottom layer of a porousnetwork of cross-linked collagen and glycosamino-glycan. The rationale for the top silicone elastomer isto control bacterial ingress and water evaporation andprovide additional mechanical support. The bottomlayer is designed to ensure rapid wound adherence.Optimal porosity of this dermal analogue allows forits slow biodegration and the induction of vascularand cellular ingrowth that eventually replaces thedermal matrix with neodermis. Several weeks later,when fibrovascular ingrowth of the dermal analoguehas occurred, the epidermal silicone layer must beremoved in the operating room and the wound closedwith a thin sheet of autologous skin [4,22,55]. Clin-ical studies with this material have been successful[21,49] and were approved by the FDA in 1997.Dermagraft (Advanced Tissue Science Inc, LaJolla, CA) [15,38] is another dermal substitute thatuses a resorbable matrix material that is similar tomaterials used in suture fabrication. This material iscomposed of a polylactin acid on which dermalfibroblasts are incorporated. Once incorporated withinthe matrix, the fibroblasts secrete growth factors andextracellular matrix proteins.A dermal allograft harvested from a living orcadaveric donor is another possible dermal substitute.Unlike the rejection of allogeneic keratinocytes ontransplantation, allografts of dermis seem to be trans-plantable without significant rejection because ofcomparatively low immunogenicity of the dermalcomponents. If rejection occurs, it is usually directedtoward the passenger leukocytes and endothelial cellsthat line the blood vessels. The use of a de-epider-mized and decellularized dermis further diminishesthe allograft immunogenicity. Allogeneic, acellulardermis prepared this way retains the structural archi-tecture of the remaining dermal matrix. This dermalmatrix has been shown to support fibroblast in-growth, neovascularization, and keratinoctye migra-tion from an overlying split-thickness skin graft orfrom seeded cultured keratinocytes [29]. A producton the market, AlloDerm (LifeCell Corp, Branch-burg, NJ), is an acellular, nonimmunogenic dermisthat retains the extracellular matrix structure and anundamaged basement membrane complex. It alsopossesses an intact vascular channel network thatallows ingrowth of fibroblasts and endothelial cellsfrom the underlying tissue.Bilayers of epithelium and dermisIn contrast to the materials science and engineeringapproach of Burke and Yannas et al. [4,22,55], Bellet al. [2] took the approach of reconstituting dermalwounds by applying a preformed tissue. They startedwith the observation that fibroblasts introduced into acollagen gel would proliferate and reorganize thecollagen into a contracted matrix containing exogen-ous collagen and the collagen and matrix proteinsproduced by the introduced fibroblasts. The rate andfinal extent of contraction varied inversely with theprotein concentration and directly with cell numberintroduced into the gel. The resulting product isdescribed as a dermal equivalent, which, unlikeK. Izumi, S.E. Feinberg / Oral Maxillofacial Surg Clin N Am 14 (2002) 61–71 63
  4. 4. Integra, relies on living cells in tissue culture toorganize the collagen network. The exact fiber struc-ture and its relationship to normal dermis are notknown. Subsequent experiments demonstrated inanimals that these collagen gels reorganized by fibro-blasts could be grafted onto full-thickness injuriesand that they would support the growth of keratino-cytes into an epidermal equivalent. The dermal com-ponent is composed of type I bovine collagen that hasbeen organized by introduction of human fibroblasts.Foreskin keratinocytes were seeded onto the surfaceof the dermal equivalent. After several days of sub-merged culturing of the skin equivalents, cultures arethen air-exposed to allow the epidermis to stratify,differentiate, and form a cornified layer [17,43]. Thetotal manufacturing period is approximately 20 days.To date, clinical evaluation of this type of skin equiv-alent has not been reported in burn patients, althoughseveral in vivo animal studies have been conducted[35]. A bilayered human skin equivalent, Apligraf(Novartis Pharmaceuticals Corp, East Hanover, NJ),already has been approved by the FDA for venousulcers and is likely to be commercially available forburn wounds.Oral mucosa substitutesPreprosthetic and reconstructive oral and maxillo-facial surgical procedures often produce open woundsin the oral mucosa. These wounds should be coveredby a graft to prevent microbial infection, excessivefluid loss, foreign material contamination, or relapse(secondary to wound contracture) and assist in theprosthetic reconstruction of the patient and in thepromotion of wound healing [13]. Currently, oralmucosa or skin grafts are used for this purpose;however, both of these grafts require a second sur-gical procedure and have disadvantages in intraoraluse [34]. Oral mucosa is an excellent intraoral graftmaterial but is available in a limited supply [31,34].Split-thickness skin grafts are available in amplesupply but may contain adnexal structures, and theyexpress a different pattern of surface keratinizationthat can lead to the development of abnormal tissuetexture in the oral cavity that could interfere withfunction [12,34,36]. The elective nature of most oraland maxillofacial surgical procedures should allowthe flexibility and timing to develop an ex vivo tissueengineered oral mucosa that could be used for intra-oral grafting procedures. The recent developments oforal keratinocyte culture techniques have paralleledthose of skin keratinocytes [24], which has enabledthe development of tissue-engineered autogenous oralmucosa that is suitable for intraoral reconstructiveprocedures [25].Structural and functional differences between skinand oral mucosaThe wet environment of the oral cavity compli-cates reconstruction with skin grafts. The keratinizedsurface of grafted skin tends to macerate and becomeeasily infected. Oral mucosa is different from skin inthat it has a moist surface and lacks adnexal struc-tures such as hair and glandular elements. Graftingof skin into the oral cavity can be complicated by thepresence of adnexal structures, which can be seen ashair growth within the mouth. Oral mucosa, unlikeskin, presents three structural variations that arelocated in specific anatomic locations within themouth. These layers are (1) masticatory mucosa(ortho or parakeratinized; hard palate, attached gin-giva), (2) lining or alveolar mucosa (nonkeratinized;lip, floor of mouth, cheek), and (3) specializedmucosa (taste buds; dorsal surface of tongue). Thekeratinized and nonkeratinized mucosa differ inthe composition of their cell layers. In keratinizedmucosa, the suprabasal cell layer is divided intothree layers and designated spinous cells, granularcells, and keratinized layers with the major cytoskel-eton keratin of 1/10 [28]. Typical keratinized mucosapossesses ‘‘keratohyalin granules’’ in the granularcell layer. Tonofibrils, aggregates of keratin fila-ments, frequently are seen in the cellular cytoplasm.In contrast, the suprabasal layer of nonkeratinizedmucosa is less evident and ordered than that seen inkeratinized mucosa. The layers are designated asspinous cell, intermediate cell, and surface cell layer,in which the major intermediate filament of keratinis 4/13. Neither keratohyalin granules nor promi-nent aggregates of keratin are seen in nonkeratin-ized mucosa.In vitro culturing techniquesMost investigators and maxillofacial surgeonshave used an irradiated layer of a transformed 3T3fibroblastic cell line as a feeder layer to propagateand expand their oral keratinocyte population togenerate oral keratinocyte (epithelial) sheets forintraoral grafting [11,45,52,54]. The oral mucosaepithelial sheet grafts were placed onto the peri-osteum of the labial aspect of the anterior mandibleto assist in performing a vestibuloplasty. All of thestudies demonstrated successful clinical outcomesand histologic findings of postgrafting biopsies, thelongest of which was 4 months postoperatively.K. Izumi, S.E. Feinberg / Oral Maxillofacial Surg Clin N Am 14 (2002) 61–7164
  5. 5. Normal epithelial layer was regenerated on the graftsites. Hata et al. [21] and Ueda et al. [53] reportedthat oral mucosa keratinocytes grew more rapidlyand differentiated less than skin keratinocytes. Der-mal substitutes for burn injuries also have beenapplied into the oral cavity, such as the bilayeredmembranes with a collagen-GAG/silastic sheet, sim-ilar to the Burke and Yannas’ Integra [4].Although they showed successful postoperativeappearances and an advantage of easy sterilizationand cost effectiveness, in the authors’ clinical experi-ence this material is difficult to handle. The silasticsheet does not present a problem with handling, butthe collagen sponge becomes ‘‘sticky’’ when itabsorbs blood, which results in difficulty duringsuturing. The environment of the oral cavity, a moistarea laden with bacteria and lytic enzymes, may notbe conducive to the collagen-rich dermal componentsused in skin equivalents. An oral mucosa equivalentnot only must be anatomically similar to mucosa butalso must possess the mechanical and handling char-acteristics of the mucosa to be useful within theintricate confines of the oral cavity.There have been reports of ‘‘oral mucosa’’ equi-valent-like Apligraf [40,41]. So far, these equivalentsare still experimental and have not been used inclinical studies. Another type of ‘‘oral mucosa’’equivalent composed of de-epidermized dermis andcultured oral mucosa keratinocytes from buccalmucosa and hard palate was studied in Korea [6,7].These oral mucosal substitutes were developed fortoxicologic and pharmacologic studies and not foruse in a clinical setting. Studies have shown that theconcurrent grafting of a dermal component aids inenhancing the quality and time of wound healing[26,32,33]. Parenteau et al. [43] showed that the rateof closure of the wound and the increase in thepercentage of wound repair is enhanced with thepresence of dermis. The maturation process and bio-logic events of skin regeneration also are acceleratedwith the presence of a dermal substrate [9]. Inokuchiet al. [23] have found that autogenous fibroblastswithin the grafted dermal matrix facilitated the long-term maintenance of the reorganized cultured epi-dermis by supporting self-renewal of the epitheliumin vivo. Clugston et al. [8] noted that the absence of agrafted dermis resulted in a contracture of culturedkeratinocyte autografts on the order of 50%.The development and grafting of a full-thicknessoral mucosal graft with a dermis can assist in epithe-lial graft adherence, minimize wound contraction,and assist in epithelial maturation while encouragingthe formation of a basement membrane [18]. Augeret al. [1] showed that a dermal equivalent would bebest made out of human, rather than animal, collagen.The human collagen (dermis) helps to promotedeposition of additional basement membrane constit-uents, which results in a more optimal pattern ofkeratinocyte differentiation and less immunogenicitythan animal collagen. The authors have been success-ful in their own laboratory in the ex vivo productionof an oral mucosal equivalent (EVPOME) using oralkeratinocytes seeded onto a human cadaver dermalmatrix, AlloDerm [24,25]. AlloDerm is an acellular,biocompatible, human connective tissue matrix withan unaltered extracellular matrix and intact basementmembrane, which consistently integrates into the hosttissue. Most importantly, AlloDerm trims, adapts, andsutures like autologous tissue. Human de-epidermizeddermis that has retained its basal lamina, consistingof keratinocytes combined with a mesenchymal ordermal component, has successfully shown enhancedepithelial morphogenesis and an increase in expres-sion of differentiation markers when it is grown at anair-liquid interface [46].Tissue-engineered oral mucosaMost reconstructive procedures in oral and max-illofacial surgery are of an elective nature. This givessurgeons the ability to time the biopsy of autogenousmucosa with the need of a sufficient size of tissuenecessary for the planned surgical reconstruction. Indeveloping a methodology to engineer any tissue, it isnecessary to abide by the requirements and restric-tions imposed by the FDA. The cultivating techniqueof Rheinwald and Green [47] uses a xenogeneicirradiated fibroblast cell line, 3T3, as a feeder layerto enhance keratinocyte growth. During the culturingperiod to expand human cells they are exposed to atransformed murine cell line. This contact potentiallycould contribute to cross-examination or transfectionof the mutational or xenogeneic DNA into the cocul-tured human keratinocytes. Serum and a xenogeneicfeeder layer contain undefined factors such as slowviruses (Creutzfeld-Jakob disease, ‘‘mad cow’’ dis-ease, or bovine spongiform encephalopathy) andforeign contaminants [50]. The importance of notusing a feeder layer and serum to culture oral mucosalautografts is obvious, especially in elective surgerybecause of the potential of the introduction of unde-termined risks to the patient. Other investigators alsosupport this point [14,27,43].In our approach to tissue engineering an oralmucosal equivalent we use a serum-free culturesystem without a feeder layer. We also have success-fully eliminated the use of bovine pituitary extractin the medium, thus having a completely definedK. Izumi, S.E. Feinberg / Oral Maxillofacial Surg Clin N Am 14 (2002) 61–71 65
  6. 6. K. Izumi, S.E. Feinberg / Oral Maxillofacial Surg Clin N Am 14 (2002) 61–7166
  7. 7. culture medium for the manufacture of their exvivo produced oral mucosal equivalent (EVPOME)[24,25]. The authors’ EVPOME is composed ofautogenous oral keratinocytes and a cadaver acellu-lar, AlloDerm (Fig. 1 A–C). Electron microscopicevaluation of the EVPOME shows that the AlloDermretains an intact basement membrane and anchoringfibrils on the papillary surface [29]. After beingcultured 4 days submerged, the authors’ EVPOMEshows several layers of keratinocytes adherent toone another via desmosomal attachments (Fig. 2),whereas specific junctional structures between basalcells and the basement membrane of the AlloDermwere not seen at that time (Fig. 3). At day 11EVPOMEs, cultured 4 days submerged and 7 daysat an air-liquid interface, numerous rudimentaryhemidesmosome-like structures were seen incorpo-rated into anchoring fibrils of the basement mem-brane of the AlloDerm (Fig. 4). This finding seems toindicate that the basal cell layer was attached firmlyto the underlying dermal equivalent of the day 11EVPOME, suggesting an ability of the epitheliallayer to withstanding shear stress.From a 4 Â 4 mm2punch biopsy of the palate itwould take approximately 40 days to fabricate anEVPOME the size of one US dollar bill. This sizeEVPOME should be large enough to cover mostmucosal defects. Approved human clinical trialswere initiated in the Fall of 2000 at the DentalSchool Hospital of Niigata University, Niigata City,Japan. Our group at the University of Michiganalso is in the process of obtaining FDA approvalfor a tissue-engineered oral mucosa for use in humanclinical trials.The clinical protocol that was used for the firstpatients in the study performed at Niigata Universityin Japan was first to take a 5 Â 5 mm2punch biopsyof the retromolar trigonal mucosa in an outpatientsetting under local anesthesia. The biopsy is plannedsufficiently before the surgical procedure to ensurethat an adequate piece of EVPOME is available forgrafting. In most cases, to date, a period of 4 weekshas been sufficient. Oral keratinocytes are dissociatedfrom the biopsy and expanded in a standard, serum-free defined culture medium. Once a sufficient num-ber of oral keratinocytes has been harvested, 1.25 ÂFig. 2. Transmission electron micrograph of keratinocytes in D4E. Numerous desmosomes (arrows) are formed betweenkeratinocytes, while abundant tonofibrils are seen in the cytoplasm of the keratinocytes (osmium tetroxide postfixation anduranyl acetate/lead citrate, original magnification Â17.000).Fig. 1. (A) Ex vivo produced oral mucosa equivalent (EVPOME) cultured 4 days submerged (D4E). Continuous epithelialmonolayer has developed over dermal component, AlloDerm (Life Cell Corporation, Branchberg, NJ; H&E, originalmagnification Âl25). (B) EVPOME cultured 4 days submerged and 7 days at an air-liquid interface (D11E). Epithelial layer ofD4E has started to stratify and differentiate. Keratinocytes in superficial layer are flattened and eosinophilic (H&E staining,original magnification Â250). (C) EVPOME cultured 4 days submerged and 14 days at an air-liquid interface (D18E). Anincrease in stratification of the layers is noted that is consistent with a more fully differentiated epithelium. Epithelial layerdemonstrates parakeratinization (H&E, original magnification Â150).K. Izumi, S.E. Feinberg / Oral Maxillofacial Surg Clin N Am 14 (2002) 61–71 67
  8. 8. 105cells/cm2are seeded onto the acellular cadaverdermal equivalent, AlloDerm. The protocol outlinedby Izumi et al. [25] is then followed. Briefly, thecomposites of oral keratinocytes and AlloDerm arecultured submerged for 4 days and at an air-liquidinterface for 7 days to encourage epithelial stratifica-tion (Fig. 1 B). This protocol was determined to beoptimal through in vivo grafting studies performed inSCID mice (Izumi et al., Tissue engineering, 2002,manuscript accepted for publication).In patients, the EVPOME is produced and trans-planted on day 11 after the oral keratinocytes havebeen seeded onto the AlloDerm. A gauze bolster orstent is then used to stabilize the EVPOMEs at thetime of surgery. Surgical stents or bolsters areremoved at 6 days postoperatively, and the surfaceof the transplanted EVPOME at the time is scrapedwith a swab for cytologic examination. The presenceof small, round-shape cells suggests the presence ofbasal cell-like characteristics. Transnasal feeding isFig. 4. Transmission electron micrograph of dermal-epithelial junction in D11E. Hemidesmosomal-like structures (arrows)incorporated into anchoring fibrils are well developed. Note anchoring fibrils (arrowheads) newly integrated within thehemidesomosomal-like structures (osmium tetroxide postfixation and uranyl acetate/lead citrate, original magnification Â30.000).Fig. 3. Transmission electron micrograph of dermal-epithelial junction in D4E. There are no specific junctional apparatus seenbetween the basal cells and basement membrane. Original, retained, anchoring fibrils (arrowheads) in the papillary surface ofAlloDerm (osmium tetroxide postfixation and uranyl acetate/lead citrate, original magnification Â17.000).K. Izumi, S.E. Feinberg / Oral Maxillofacial Surg Clin N Am 14 (2002) 61–7168
  9. 9. used until removal of the stitches to minimize disrup-tion of the grafted EVPOME. A soft diet is begun atpostoperative day 8 at the time of removal of thetransnasal feeding tube. At 4 weeks postoperatively, apunch biopsy is performed for histologic examination.In some cases, AlloDerm without autogenous ker-atinocytes as a control also has been transplanted ontooral mucosal defects. In contrast to the EVPOME,the AlloDerm graft without an epithelium showedmore shrinkage over time postoperatively, whichresulted in a greater degree of wound contraction.The AlloDerm graft without an epithelium caused anindurated wound, which could impair soft tissuemobility. On histopathologic examination of 4 weeksafter surgery, the epithelial layers of the EVPOMEand AlloDerm without epithelium demonstrated aregenerative, well-stratified epithelial layer. The pres-ence of endothelial cells was evident as was a markedvascular ingrowth and cellular infiltration into theunderlying dermal component of the EVPOME andAlloDerm alone. Because the presence of the forma-tion of intense granulation tissue may lead to addi-tional scarring, the grafted AlloDerm without anepithelium might result in a functional compromisewithin the oral cavity. The histopathologic features ofgrafted EVPOMEs showed a favorable remodelingand incorporation within the host tissue during thehealing phase.Studies are in progress using tissue-engineeredoral mucosa as a vehicle for the use of gene therapyto enhance wound healing and/or transmucosallyadminister systemically needed growth factors.SummaryTo date, successfully developed EVPOMEs in aserum-free culture system without a feeder layer arethe most acceptable and promising oral mucosalsubstitutes for human intraoral grafting because ofminimal risk of foreign contaminants, easy handlingand stitching, subsequent rapid revascularization intodermal component after transplantation, and contri-bution to favorable open wound closure withoutfunctional compromise, although several types of oralmucosal substitutes described in this article have beenused in patients.AcknowledgmentThe authors thank Masaaki Hoshino for histechnical assistance, Dr. Michiko Yoshizawa for herinput and involvement in the development of ourtissue-engineered human oral mucosa, and Dr.Cynthia Marcelo for many fruitful discussions.References[1] Auger FA, Lopez Valle CA, Guignard R, Tremblay N,Noel B, Goulet F, et al. Skin equivalent produced withhuman collagen. In Vitro Cell Dev Biol Anim 1995;31:432–9.[2] Bell E, Ehrlich HP, Buttle DJ, Nakatsuji T. Livingtissue formed in vitro and accepted as skin-equivalenttissue of full thickness. Science 1981;211:1052–4.[3] Boyce ST, Supp AP, Harriger MD, Pickens WL, Wick-ett RR, Hoath SB. Surface electrical capacitance as anoninvasive index of epidermal barrier in cultured bar-rier in cultured skin substitutes in athymic mice. J In-vest Dermatol 1996;107:82–7.[4] Burke JF, Yannas IV, Quinby WC Jr, Bondoc CC, JungWK. Successful use of a physiologically acceptableartificial skin in the treatment of extensive burn injury.Ann Surg 1981;194:413–28.[5] Carter DM, Lin AN, Varghese MC, Caldwell D, PrattLA, Eisinger M. Treatment of junctional epidermolysisbullosa with epidermal autografts. J Am Acad Derma-tol 1987;17:246–50.[6] Cho KH, Ahn HT, Park KC, Chung JH, Kim SW, SungMW, et al. Reconstruction of human hard-palate mu-cosal epithelium on de-epidermized dermis. J DermatolSci 2000;22:117–24.[7] Chung JH, Cho KH, Lee DY, Kwon OS, Sung MW,Kim KH, et al. Human oral buccal mucosa recon-structed on dermal substrates: a model for oral epi-thelial differentiation. Arch Dermatol Res 1997;289:677–85.[8] Clugston PA, Snelling CF, MacDonald IB, Maledy HL,Boyle JC, Germann E, et al. Cultured epithelial auto-grafts: three years of clinical experience with eighteenpatients. J Burn Care Rehabil 1991;12:533–9.[9] Compton CC. Acceleration of skin regeneration fromcultured epithelial autografts by transplantation tohomograft dermis. J Burn Care Rehabil 1993;14:653–62.[10] Cuono CB, Langdon R, Birchall N, Barttelbort S,McGuire J. Composite autologous allogeneic skin re-placement: development and clinical application. PlastReconstr Surg 1987;80:625–35.[11] De Luca M, Albanese E, Megna M, Cancedda R, Man-giante PE, Cadoni A, et al. Evidence that human oralepithelium reconstituted in vitro and transplanted ontopatients with defects in the oral mucosa retains proper-ties of the original donor site. Transplantation 1990;50:454–9.[12] Dellon AL, Tarpley TM, Chretien P. Histologic evalu-ation of intraoral skin grafts and pedicled flaps in hu-mans. J Oral Surg 1976;34:789–94.[13] Donoff RB. Biological basis for vestibuloplasty proce-dures. J Oral Surg 1976;34:890–6.K. Izumi, S.E. Feinberg / Oral Maxillofacial Surg Clin N Am 14 (2002) 61–71 69
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