Intraoral Wound Closure with Tissue- Engineered Mucosa ... - CiteSeerX [PDF]

Intraoral Wound Closure with TissueEngineered Mucosa: New Perspectives for Urethra Reconstruction with Buccal Mucosa Grafts Günter Lauer, M.D., D.M.D., Ph.D., Ronald Schimming, M.D., D.M.D., and Alexander Frankenschmidt, M.D., Ph.D. Freiburg, Germany

closure with local tissue, the technique presented in this study reduces the severity of postoperative pain and allows faster rehabilitation in patients because of a better woundhealing process. Furthermore, better mobility of intraoral soft tissue structures is achieved. (Plast. Reconstr. Surg. 107: 25, 2001.)

In urethra reconstruction, the creation of a new urethra from a free oral mucosa graft is an established surgical technique. The oral mucosa is removed at the same time that the urethra reconstruction procedure is performed. Depending on the size of graft required, the intraoral wound is closed primarily or left to heal secondarily. The latter method limits this technique by leading to scars or strictures, which have a negative impact on the condition of the intraoral soft tissue. Therefore, in this study, a pilot study involving 12 patients, tissue-engineered mucosa was tested for covering intraoral defects to avoid the drawbacks mentioned above. For mucosa tissue-graft engineering, a biopsy sample 2 to 4 mm in diameter was removed from the hard palate approximately 4 weeks before the urethra reconstruction procedure was to be performed. In addition, 30 ml of autogenous serum was extracted from a venous whole-blood sample. The primary cultures were incubated in Dulbecco modified Eagle’s medium and nutrient factor F 12 (Gibco Co., Eggenstein, Germany), containing the usual additives and autogenous serum. After a period of 3 weeks, subcultivation was performed to engineer mucosa transplants consisting of several layers of keratinocytes on a support foil. After thorough intraoperative blood coagulation had occurred, the cultured mucosa graft on the carrier foil was applied on the wound surface and fixed by single sutures. Additionally, the cultured mucosa graft was covered for 8 to 10 days by an intraoral dressing, which was also fixed onto the wound surface by single suture loops. It is possible to perform primary intraoral wound closure with tissue-engineered mucosa to cover defect sizes as large as 11.0 ⫻ 4.0 cm. This new method provides a better prospect for both urethra reconstruction and the reconstruction of intraoral tissue defects. The number and size of intraoral scars and strictures are diminished. This is of special interest for the reconstruction of the functional unit oral cavity, including soft tissue and cosmetic conditions (e.g., in case of prosthetic rehabilitation). In comparison to primary wound

Reconstruction of the male urethra is required for a multitude of congenital anomalies, such as hypospadias and epispadias, as well for acquired lesions, such as stricture disease. Most of these patients require a free-tissue graft for creation of a neourethra. Different sources of nongenital skin have been described in the literature. Initially, full-thickness skin grafts from hair-bearing sites provided a reasonable success rate. However, over time, complications such as strictures and balanitis xerotica obliterans have occurred. 1,2 Other tissue sources have been used, including non– hairbearing skin,2– 4 the ureter, tunica vaginalis,5 and cultured urethral epithelium.6 All these sources have limitations that have restricted their widespread use. Humby and Memmelaar were among the first researchers to describe the use of bladder mucosa for urethral replacement.7,8 Although the subsequent use of bladder mucosa became quite popular, complications during its harvesting and meatal exuberance or the occurrence of cauliflower deformity as a result of exposure of the bladder

From the Department of Oral and Maxillofacial Surgery and the Department of Urology, University Clinic of Freiburg. Received for publication January 11, 2000; revised April 3, 2000. Presented at the Second BioValley Tissue Engineering Symposium, Freiburg, Germany, November 25 through 27, 1999.

25

26

mucosa at the meatus have been described.9,10 In 1941, Humby suggested the use of buccal mucosa for urethral replacement.7 However, because of the higher rate of infection involved with the use of this graft, bladder mucosa was favored.7,11 In recent decades, oral and maxillofacial surgeons have achieved good results after tumor ablation by using free buccal mucosa grafts for reconstruction of the conjunctiva and the eyelid, in sulcoplasty, and in the closure of defects of the tongue and the cheek.12–17 These grafts have also demonstrated good results in reconstructive surgery of the larynx and trachea.12,18 Such grafts are extremely elastic and show only a slight tendency to retract.18 –20 These findings confirm the experience already gathered in the field of eye surgery, in which buccal mucosa has been successfully used for a number of years to cover defects in the palpebral and bulbar conjunctiva.21 In 1992, more than 50 years after Humby, Bürger et al.22 and Dessanti et al.11 reported their first clinical experiences with urethra reconstruction with buccal mucosa grafts. Their excellent results stimulated a number of different working groups, and today, urethra reconstruction with buccal mucosa grafts represents the standard procedure.23–28 Buccal mucosa is a markedly resistant nonkeratinizing epithelium with a thin and highly vascularized lamina propria. The flexibility of the graft, ease involved in trimming it, and good long-term urethral results also add to its clinical success. Whereas urethra reconstruction profits from the use of buccal mucosa, local sequelae at the donor site— especially in cases of patients who have undergone multiple surgical procedures—may negatively influence the clinical outcome. Submucosal scarring with contracture and subsequent web formation, limitation of mandibular movement, and injuries of the parotid duct with decreased parotid salivary flow may impact the success of the procedure and result in further surgery (e.g., to release mucosal tension).29 –31 These possible complications limit the buccal mucosa graft size to 3 ⫻ 7 cm and can be of paramount importance in cases where multiple reconstruction procedures including buccal mucosa grafts have already been performed. In this study, to avoid these complications and provide alternate therapy concepts for the classes of patients mentioned above, the application of tissue-engineered oral mucosa for

PLASTIC AND RECONSTRUCTIVE SURGERY,

January 2001

covering donor-side defects was discussed and a prospective study was initiated. PATIENTS

AND

METHODS

Patients

Between April of 1997 and June of 1998, 12 patients received buccal mucosa grafts for urethral reconstructions with subsequent intraoral wound closure with cultured mucosa. Patients’ ages ranged from 2 to 30 years (Table I). Nine patients were referred after several unsuccessful attempts at hypospadias repair. Of the 12 repairs performed, all were one-stage procedures performed while the patient was under general anesthesia, with simultaneous harvesting of buccal mucosa grafts after penile dissection, including chordee and stricture excision. The graft sizes are listed in Table I. Tissue Engineering

To engineer mucosal tissue grafts, a biopsy sample approximately 2 to 4 mm in diameter was removed from the hard palate approximately 4 weeks before the graft operation was performed. In addition, 30 ml of autogenous serum was extracted from venous whole-blood samples. In patients 1 and 2 (who were both infants), the venous whole-blood sample was drawn from the parents. Under conditions of good manufacturing, the mucosal keratinocyte grafts were cultured by use of a previously described explant technique.32 The primary cultures were incubated in Dulbecco modified Eagle’s medium and nutrient factor F 12 (in a 3:1 ratio) containing the usual additives and autogenous serum.33 After a period of 18 days, and 3 days before the graft was implanted, subcultures of mucosa keratinocytes were seeded on Tissue foil (Baxter Immuno Co., TABLE I Graft Sizes and Patients’ Ages (n ⫽ 12) Patient No.

Age (years)

Graft Size (cm)

1 2 3 4 5 6 7 8 9 10 11 12

2 2 12 15 17 23 23 26 29 29 30 30

2.0 ⫻ 4.0 2.0 ⫻ 5.0 2.5 ⫻ 9.0 2.5 ⫻ 7.0 4.0 ⫻ 11.0 3.0 ⫻ 10.0 3.0 ⫻ 9.0 4.0 ⫻ 10.0 2.5 ⫻ 9.0 4.0 ⫻ 11.0 3.5 ⫻ 10.0 4.0 ⫻ 11.0

Vol. 107, No. 1 /

27

TISSUE ENGINEERING IN ORAL WOUND REPAIR

TABLE II Maximal Incisal Mouth Opening with Time

Patient No.

Preoperative Measurement (mm)

1 Day

1 Week

2 Weeks

6 Weeks

6 Months

1 2 3 4 5 6 7 8 9 10 11 12

32 34 51 47 54 53 42 45 52 48 42 51

25 25 26 39 40 38 32 41 41 37 36 44

29 28 35 45 44 45 39 45 47 42 42 46

32 33 39 45 48 48 40 44 49 46 42 49

32 34 50 47 54 52 42 45 52 48 43 51

32 34 50 47 54 52 42 45 52 48 43 51

Postoperative Measurement (mm)

Heidelberg, Germany), a collagen foil used in mucosa-transplant engineering (Fig. 1). Buccal Mucosa Grafting

A full-thickness mucosal graft measuring no larger than 4 ⫻ 11 cm was taken from the buccal plane area between the parotid duct and inferior vestibular fold and extending in an anterior direction into the lower lip. A solution of one part epinephrine to 100,000 parts 1% lidocaine was injected submucosally at the incision side for better hemostasis. An average of 20-percent shrinkage had to be taken into account before the graft was outlined. Careful dissection prevented any damage to the underlying mental nerve and oral musculature. The undersurface of the mucosal graft was defatted and a tube formed before anastomosis to the urethra was performed (Fig. 2).

After the intraoral blood vessels thoroughly coagulated, the cultured mucosa graft on the collagen foil was laid on the wound surface and secured by single suture loops (4-0 Vicryl) to the adjacent mucosa (Fig. 3). Additionally, the cultured mucosa graft was covered by an intraoral gauze dressing containing disinfectant solution (Iodoform-Glycerol, Jena-pharm Inc., Jena, Germany) for 8 to 10 days; this dressing had also been fixed to the wound surface by single suture loops, similar to a tie-over (Fig. 4). All patients were fed by mouth beginning on day 1 after surgery. Patients 1 and 2 initially had to be fed with intravenous liquid nutritional substitutions for 2 days after surgery. Oral disinfection and cleansing with chlorinated water (Chlorhexamed, Bayer Inc., Leverkusen, Germany) was performed daily. This procedure continued after the removal of the tie-over and until the formation of a complete mucosal layer was observed. Postoperative Assessment

The healing of the cultured graft was clinically, histologically, and immunohistologically assessed. Clinical Assessment

FIG. 1. Tissue-engineered mucosa graft.

During the early postoperative healing period (up to day 30), wound healing, and especially the epithelialization, was observed closely (every other day). During the further postoperative observation interval of up to 25 months, patients’ clinical situations were recorded. Because the buccal mucosa was topographically difficult, repeated measurements of the grafted area could not be performed, but the mobility of the cheek, opening of the mouth,

28

FIG. 2. Situation after the buccal mucosal graft was defatted and the urethral tube formed.

PLASTIC AND RECONSTRUCTIVE SURGERY,

January 2001

70% alcohol, further dehydrated, and embedded in Technovit 8100 (Kulzer Co., Wehrheim, Germany). Semithin sections were cut and stained with hematoxylin and eosin. For immunohistological assessments, the methylmethacrylate semithin sections were predigested with 0.1% trypsin solution, and after being rinsed with saline, the unspecific bindings were blocked with 10% horse serum. The immunohistochemical protocol using the Vectastain (Vector Laboratories, Burlingame, Mass.) method was performed, incubating the primary and secondary antibodies for 30 minutes each. The antibodies for cytokeratins 5/6, 13, 14, and 17 (Boehringer Co., Mannheim, Germany), laminin, and collagen IV (Sigma Co., Munich, Germany) were used. The slides were examined using a Zeiss Axiolab microscope (Zeiss Co., Munich, Germany) at ⫻10 and ⫻25 magnification. The ethics commission of the Faculty of Medicine approved the procedure (procedure no. 94/99, ethics commission, Faculty of Medicine, University of Freiburg, Germany). RESULTS

FIG. 3. Clinical situation after the foil with cultured mucosa was applied and fixed with suture loops.

FIG. 4. The transplanted mucosa covered with gauze dressing.

and neurosensory investigation of the buccal and lower lip mucosa could be assessed. Histological and Immunohistological Assessments

Biopsy specimens were taken from the grafted sites between day 8 and 12 months after the operation. The specimen were fixed in

At the time the intraoral wound dressings were removed, usually 8 to 10 days after the operation, an epithelialized but still vulnerable wound surface covered with little fibrin was observed in all patients (Fig. 5). The wound surfaces stabilized during the next days. In all patients, a complete mucosal layer had formed, on average, by day 16 after the operation. At the clinical follow-up visit 1 month after the operation, each patient displayed a mucosal layer with minimal superficial scar formation. After 3 months, no further clinical differentiation between local mucosa (control side) and grafted mucosa could be described (Fig. 6). No scar formation was found. Further clinical follow-up visits after 6, 9, and 12 months—and, in five cases, after 25 months— confirmed the 3-month findings. During the first few postoperative days, all patients had moderate intraoral discomfort, which resolved by 16 days after the operation. The results for maximal incisal opening measurements are listed in Table I. A measurement equal to the preoperative maximal incisal opening was achieved after an average time of between 2 and 6 weeks after the operations. A deviation to the wound site of approximately 3 mm accompanied the initial limitation of mouth-opening. The deviation disappeared

Vol. 107, No. 1 /

29

TISSUE ENGINEERING IN ORAL WOUND REPAIR

FIG. 5. Intraoral clinical situation after 10 days: the epithelialized but still vulnerable wound surface covered with little fibrin.

FIG. 6. Situation after 3 months, with no clinical differentiation between local mucosa (control side, left) and grafted mucosa (right).

with the increase in the degree of mouthopening. No patients had temporomandibular joint disorders or other related myopathic problems accompanied by the mouth-opening limitation. No patients described any symptoms of dry mouth after the operation, and clinical examinations showed a normal salivary flow from the ipsilateral parotid duct 1 week after the operations in all cases. No patient described neurosensory disturbances of the ipsilateral buccal or lower lip area. Subjective assessment was confirmed by use of the Pain and Thermal Sensitivity Tester (PATH-test, Phywe Co., Munich, Germany). The histological findings on day 8 at the time the dressing was removed in each patient showed conglomerates of keratinocytes with positive immunostaining for cytokeratins 5/6 and 17, surrounded by edematous extracellu-

lar matrix–rich connective tissue at the upper side of the wound (Fig. 7). The basal border of those keratinocyte conglomerates and parts of the basal membrane were rendered immunohistochemically visible by the reaction of anticollagen IV and antilaminin in dotted patterns (Fig. 8). By postoperative day 16, a multilayered epithelium had formed. The keratinocytes of all layers expressed cytokeratins 14 and 17. The mucosal epithelium rested on a complete continuous basal membrane that was positive for collagen IV and laminin in linear patterns. Four to eight weeks after the operation, a multilayered epithelium without differentiation into strata was visible. There were 20 to 40 cell layers. Cytokeratins 5/6, 14, and 17 were positive in all layers. Twenty weeks after the operation, some stratification was visible, but all layers still stained positive for cytokeratins 5/6 and 14 (Fig. 9). In the later postoperative observation period, the histologic configuration of the grafted mucosa changed to a normal appearance. Six months after the operation and more, a differentiated mucosal epithelium was observed. The different strata could be distinguished (basal stratum, spinous stratum, stratum distendum) (Fig. 10). The cell differentiation as judged by the cytokeratin staining resembled that of nongrafted alveolar mucosa. Cytokeratins 5/6 were primarily expressed in the basal and adjacent suprabasal layer, whereas the cytokeratin 13 reaction was predominantly restricted to all upper strata, except the basal cells. This result was also found in biopsies taken at a later postoperative stage in three patients (at 25 months). DISCUSSION

The use of a free buccal mucosa graft to repair hypospadias has several advantages, including flexibility, readily available graft material, one-stage correction of even the most serious defects, and good cosmetic reconstruction. In comparison with other grafts (e.g., bladder mucosa), this graft provides better long-term results and can reduce the complication rate.3,11,23 Despite the good long-term results in urethra reconstruction, intraoral donor-site morbidity was described in several cases, which may limit this graft-harvesting technique.29 –31 Submucosal scarring and injuries of the parotid duct can especially initiate

30

PLASTIC AND RECONSTRUCTIVE SURGERY,

FIG. 7. Immunohistology for cytokeratin 17 in a biopsy sample taken 8 days after the graft was placed. Conglomerates of cytokeratin 17–positive cells (arrows) are evident in an edematous matrix. CT, connective tissue at the basal aspects of the conglomerates. (Methylene blue stain, original magnification ⫻100.)

further complications, including limited mouth opening, myoarthropathy, and dry mouth. Reduction of the buccal vestibule may impact prosthetic rehabilitation and can require additional surgical corrections. Thus the

January 2001

size of the buccal mucosa graft is limited. This is of utmost importance in patients who have already undergone several previous procedures and for whom there is no alternative to the use of buccal mucosa grafts. In such cases, large grafts are often necessary for sufficient reconstruction. To avoid the complications mentioned above, several techniques for intraoral wound closure have been discussed. Beside primary wound closure, which is feasible only for small defects19,31,34 without later distortion of the parotid duct, secondary wound healing35,36 and temporary covering with Lyodura, Syspurderm, or Ethisorp37 have been described. Secondary wound healing often leads to extended postoperative discomfort for the patient and provides scar formation. Temporary wound closure allows secondary wound healing under a dressing. No long-term complications for this technique were outlined, although a higher rate of patient discomfort in the early postoperative period was noted.37 From a clinical point of view, reconstruction of intraoral mucosal defects with local mucosa will provide the best long-term results. This investigation provides evidence that tissueengineered mucosa can well serve as grafts for covering intraoral wounds caused by the harvesting of buccal mucosa transplants in patients treated for urethra reconstruction. Only a small intraoral biopsy (maximum size, 4 mm diameter) is necessary, and the biopsy wound is closed primarily. Furthermore, an adequate

FIG. 8. Immunostaining for collagen IV in a biopsy sample taken 8 days after the graft was placed. There is a dotted staining pattern at the basal aspect of the keratinocyte conglomerates (arrows). V, vessel in the connective tissue. (Methylene blue stain, original magnification ⫻300.)

Vol. 107, No. 1 /

TISSUE ENGINEERING IN ORAL WOUND REPAIR

31

FIG. 9. Immunostaining for cytokeratin 14 in a biopsy sample taken 20 weeks after the graft was placed. There is some stratification in the healed transplanted tissue-engineered mucosa grafts. The differentiation marker for the basal cell cytokeratin 14 is still present in all cell layers. (Methylene blue stain, original magnification ⫻100.)

FIG. 10. Histological specimen with normal differentiated mucosal epithelium in a biopsy sample taken 6 months after the graft was placed. Stratification (basal stratum, spinous stratum, stratum distendum) is well established. (Hematoxylin and eosin stain, original magnification ⫻100.)

postoperative period (approximately 16 days), not an extended interval for the wound healing, was observed. Careful submucosal preparation and intraoral wound closure with cultured mucosa allow primary defects of up to 4 ⫻ 11 cm without long-term complications. If

necessary, a second buccal mucosa graft can be harvested in the same way from the contralateral side. This technique allows extended urethral reconstruction procedures. A differentiation into parakeratotic stratified epithelium takes place, as demonstrated histologically and immunohistologically. During the postoperative healing of the donor graft site, there is a sequence and topographic order in the appearance of extracellular and intracellular markers, which ends with a distribution pattern as described in normal alveolar mucosa.38 Similar clinical, biological, and histological results were described when skin wounds were covered with cultured keratinocyte grafts.39 In comparison to primary wound closure or secondary wound healing, tissue-engineered mucosa grafts depend on a considerable amount of preoperative work and laboratory preparations. At present, this limits the application of the method to selected cases and treatment centers that can provide adequate facilities. Furthermore, mucosal grafts require local submucosal connective tissue for uncomplicated healing procedure. Therefore, direct urethral reconstruction with engineered oral mucosa is not feasible. Further developments in tissue engineering may reduce these disadvantages: for example, when the mucosa layer can be engineered directly on the carrier material.40 The use of a collagen matrix may lead to the engineering of complete mucosa–submucosa grafts already

32

PLASTIC AND RECONSTRUCTIVE SURGERY,

described for skin layers.41 This may the allow engineering of mucosa–submucosa grafts for urethral reconstruction, thereby avoiding creation of any buccal defects. Günter Lauer, M.D., D.M.D., Ph.D. Department of Oral and Maxillofacial Surgery University Hospital Carl-Gustav-Carus Dresden Fetscher Strasse 74 Dresden, D-01307 Germany [email protected]

19.

20.

21. 22.

REFERENCES 1. Secrest, C. L., Jordan, G. H., Winslow, B. H., et al. Repair of the complications of hypospadias surgery. J. Urol. 150: 1415, 1993. 2. Webster, G. D., Brown, M. W., Koefoot, R. B., Jr., and Sihelnick, S. Suboptimal results in full-thickness skin graft urethroplasty using an extrapenile skin donor site. J. Urol. 131: 1082, 1984. 3. Hendren, W. H., and Crooks, K. K. Tubed free skin graft for construction of male urethra. J. Urol. 123: 858, 1980. 4. Vyas, P. R., Roth, D. R., and Perlmutter, A. D. Experience with free grafts in urethral reconstruction. J. Urol. 137: 471, 1987. 5. Mitchell, M. E., Adams, M. C., and Rink, R. C. Urethral replacement with ureter. J. Urol. 139: 1282, 1988. 6. Snow, B. W., and Cartwright, P. C. Tunica vaginalis urethroplasty. Urology 40: 442, 1992. 7. Humby, G. A one-stage operation for hypospadias. Br. J. Surg. 29: 84, 1941. 8. Memmelaar, J. Use of bladder mucosa in one-stage repair of hypospadias. J. Urol. 58: 68, 1947. 9. Keating, M. A., Cartwright, P. C., and Duckett, J. W. Bladder mucosa in urethral reconstructions. J. Urol. 144: 827, 1990. 10. Ransley, P. G., Duffy, P. G., Oesch, I. L., Van Oyen, P., and Hoover, D. The use of bladder mucosa and combined bladder mucosa/preputial skin grafts for urethral reconstruction. J. Urol. 138 (4 Pt. 2): 1096, 1987. 11. Dessanti, A., Rigamonti, W., Merulla, V., Falchetti, D., and Caccia, G. Autologous buccal mucosa graft for hypospadias repair: An initial report. J. Urol. 147: 1081, 1992. 12. Cole, J. G. Reconstruction of large defects of the upper eyelid. Am. J. Ophthalmol. 64: 376, 1967. 13. Campbell, C. B., III, Shannon, G. M., and Flanagan, J. C. Conjuntivodacryocystorhinostomy with mucous membrane graft. Ophthalmic Surg. 14: 647, 1983. 14. Shore, J. W., Foster, C. S., Westfall, C. T., and Rubin, P. A. Results of buccal mucosal grafting for patients with medically controlled ocular cicatricial pemphigoid. Ophthalmology 99: 383, 1992. 15. Meyer, R. The graft of buccal mucous membrane in the reconstructive cervicofacial surgery. Med. Hyg. 29: 450, 1971. 16. Fitzpatrick, B. N. Free full thickness mucosal grafts in the mouth. Aust. Dent. J. 21: 131, 1976. 17. Steinhäuser, E. W. Free transplantation of oral mucosa for improvement of denture retention. J. Oral Surg. 27: 955, 1969. 18. Matras, H. Clinical and experimental study of the trans-

23.

24.

25.

26.

27.

28. 29.

30.

31.

32.

33.

34.

35.

36.

37.

January 2001

plantation of free grafts of buccal mucous membrane inside and outside the buccal cavity. Med. Hyg. 29: 447, 1971. Morey, A. F., and McAninch, J. W. When and how to use buccal mucosal grafts in adult bulbar urethroplasty. Urology 48: 194, 1996. Wessells, H., and McAninch, J. W. Use of free grafts in urethral stricture reconstruction. J. Urol. 155: 1912, 1996. Roper-Hall, M. J. Stallard’s Eye Surgery, 6th Ed. Philadelphia: Lippincott, 1980. Pp. 125–127. Bürger, R. A., Müller, S. C., El-Damanhoury, H., Tschakaloff, A., Riedmiller, H., and Hohenfellner, R. The buccal mucosal graft for urethral reconstruction: A preliminary report. J. Urol. 147: 662, 1992. Fichtner, J., Fisch, M., Filipas, D., Thuroff, J. W., and Hohenfellner, R. Refinements in buccal mucosal grafts urethroplasty for hypospadias repair. World J. Urol. 16: 192, 1998. Kropfl, D., Tucak, A., Prlic, D., and Verweyen, A. Using buccal mucosa for urethral reconstruction in primary and re-operative surgery. Eur. Urol. 34: 216, 1998. Payne, C. E., Sumfest, J. M., and Deshon, G. E., Jr. Buccal mucosal graft for hypospadias repair. Tech. Urol. 4: 173, 1998. Caldamone, A. A., Edström, L. E., Koyle, M. A., Rabinowitz, R., and Hulbert, W. C. Buccal mucosal grafts for urethral reconstruction. Urology 51 (5A Suppl.): 15, 1998. Baskin, L. S., and Duckett, J. W. Buccal mucosa grafts in hypospadias surgery. Br. J. Urol. 76 (Suppl. 3): 23, 1995. Naude, J. H. Buccal mucosal grafts in the treatment of ureteric lesions. BJU Int. 83: 751, 1999. Neuhaus, R. W., Baylis, H. I., and Shorr, N. Complications at mucous membrane donor sites. Am. J. Ophthalmol. 93: 643, 1982. Brasher, W. J., Rees, T. D., and Boyce, W. A. Complications of free grafts of masticatory mucosa. J. Periodontol. 46: 133, 1975. Tolstunov, L., Pogrel, M. A., and McAninch, J. W. Intraoral morbidity following free buccal mucosa graft harvesting for urethroplasty. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 84: 480, 1997. Lauer, G. Autografting of feeder-cell free cultured gingival epithelium: Method and clinical application. J. Craniomaxillofac. Surg. 22: 18, 1994. Lauer, G. Autogenous serum for culturing keratinocyte autografts. In G. O. Phillips, R. von Versen, D. M. Strong, and A. Nather (Eds.), Advances in Tissue Banking, Vol. 1. Singapore: World Scientific, 1997. P. 183. Duckett, J. W., Coplen, D., Ewalt, D., and Baskin, L. S. Buccal mucosal urethral replacement. J. Urol. 153: 1660, 1995. Lopez, J. A., Valle, J., Timon, A., et al. Use of autologous buccal mucosa graft for urethral surgery in males. Eur. Urol. 29: 227, 1996. Ahmed, S., and Gough, D. C. S. Buccal mucosal graft for secondary hypospadias repair and urethral replacement. Br. J. Urol. 80: 328, 1997. Szulczewski, D. H., Kleinheinz, R., Werkmeister, R., Meyer, U., Roth, S., and Joos, U. Regeneration der Mundschleimhaut im Planum buccale nach Ent-

Vol. 107, No. 1 /

TISSUE ENGINEERING IN ORAL WOUND REPAIR

nahme zur Rekonstruktion von bulbären Urethrastenosen. Mund Kiefer Gesichtschir. 3: 34, 1999. 38. Ouhayoun, J. P., Gosselin, F., Forest, N., Winter, S., and Franke, W. W. Cytokeratin patterns of human oral epithelia: Differences in cytokeratin synthesis in gingival epithelium and adjacent alveolar mucosa. Differentiation 30: 123, 1985. 39. Compton, C. C., Gill, J. M., Bradford, D. A., Regauer, S., Gallico, G. G., and O’Connor, N. E. Skin regenerated from cultured epithelial autografts on full-thick-

33

ness burn wounds from 6 days to 5 years after grafting. Lab. Invest. 60: 600, 1989. 40. Gutwald, R., Lauer, G., Otten, J. E., and Schilli, W. Epithelzellen und Fibroblasten der Gingiva auf resorbierbaren Membranen: Gewebetransfer zur Wundheilung? D. Zahnärztl. Z. 49: 1015, 1994. 41. Hansbrough, J. F., Morgan, J., Greenleaf, G., Parikh, M., Nolte, C., and Wilkins, L. Evaluation of Graftskin composite grafts on full-thickness wounds on athymic mice. J. Burn Care Rehabil. 15: 346, 1994.