First experience using a novel microsurgical robotic device for free flap surgery in cranio- and maxillofacial surgery.

Many surgical fields profit from robotic support devices. After the first case reports about the use of a special microsurgical roboter (Symani Sugrical System, Medical Microinstruments, Pisa, Italy) we evaluated the potential of such a device in cranio- and maxillofacial surgery in a world's first single-center case series. This novel piece of equipment is meant to assist the surgeon anastomosing small vessels, nerves and lymphatic vessels. In total 30 free flaps were performed and compared to another 30 conventionally anastomosed free flaps. In total 127 anastomoses were surveyed. We encountered a lot of potential for robotic supported operations in the field of cranio- and maxillofacial surgery. However, the surgery time for robotic supported anastomosis with an average time of 32.5 min to perform arterial anastomosis was significant longer than the conventional ones, which needed 11.8 min on average. Tremor Filter and Motion Scaling are promising features for future microsurgery but the grip of the microinstruments has to be improved. It remains to be seen if the potential will be validated after the upcoming learning period and if robotic support devices will prevail in cranio- and maxillofacial surgery.

Scapular free flap as a good choice for mandibular reconstruction: 119 out of 280 cases after resection of oral squamous cell carcinoma in a single institution.

Microsurgical procedures for reconstruction after resection of head and neck tumours have become standardised and reliable. Among them, the scapular free flap is used less often, mostly to avoid excessive operating times. We hypothesise that complex reconstructions after resection of oral squamous cell carcinoma (OSCC) are successful even with time-consuming free flaps such as the scapular free flap. In this retrospective, single-centre study, we used the evaluation of medical records to investigate the postoperative outcome of microvascular reconstruction after ablative surgery of OSCC. Associations among the categorical variables were analysed using Pearson's chi squared test or Fisher's exact test. Among the continuous variables, the t test or Mann-Whitney U test were used as appropriate. For multivariate analysis, the logistic regression model was calculated. In the sample of 280 free flap reconstructions, we performed 142 radial forearm and 119 scapular free flaps. The American Society of Anesthesiology (ASA) score (p=0.006) and the duration of the operation (p=0.010) are independent factors which influence the need for operative revisions. The type of free flap is irrelevant for that. With 4.2% flap losses, scapular free flaps were successful; even in patients ≥ 70 years old (0 flap losses). Complex reconstructions after surgical resection of OSCC are successful even in aged patients. The scapular free flap is a good choice for mandibular reconstruction despite the time-consuming intraoperative repositioning of the patient. In an increasingly ageing group of patients, who have more vascular diseases, scapular free flaps could be a very successful alternative after ablative surgery of oral squamous cell carcinoma.

Salivary MMP-9 in the detection of oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most common malignant tumour of the oral cavity. Detection of OSCC is currently based on clinical oral examination combined with histopathological evaluation of a biopsy sample. Direct contact between saliva and the oral cancer makes measurement of salivary metalloproteinase-9 (MMP-9) an attractive alternative. MATERIAL AND METHODS: In total, 30 OSCC patients and 30 healthy controls were included in this prospective study. Saliva samples from both groups were collected, centrifuged and supernatant fluid was subjected to ELISA for assessment of MMP-9. The median salivary MMP-9 values with interquartile range (IQR) of OSCC patients and the control group were statistically analysed using the Mann-Whitney U-test. The receiver operating characteristic (ROC) curve was constructed and the area under curve (AUC) was computed. RESULTS: The median absorbance MMP-9 value of the OSCC group was 0.186 (IQR= 0.158) and that of control group was 0.156 (IQR=0.102). MMP-9 was significantly increased in the OSCC patients than in the controls by +19.2% (p=0.008). Median values in patients with recurrence and in patients with primary event were 0.233 (IQR=0.299) and 0.186 (IQR=0.134) respectively. MMP-9 was significantly increased in patients with primary event (p=0.017) compared to controls by +19.2%. No significant increase of MMP-9 level was detected when comparing patients with recurrence and healthy controls (+49.4%; p=0.074). The sensitivity value of MMP-9 was 100% whereas the specificity value was 26.7% with AUC of 0.698. CONCLUSIONS: The present data indicates that the elevation of salivary levels of MMP-9 may be a useful adjunctive diagnostic tool for detection of OSCC. However, further studies are necessary to provide scientific and clinical validation.

Wearing complete dental prostheses - Effects on perioral morphology.

BACKGROUND: To adequately perform rehabilitation of edentulous patients by a complete removable dental prosthesis (CRDP) is from basic interest to dentists to understand the morphologic changes caused by re-establishment of a physiologic jaw relationship. Anthropometric analyses of standardized frontal view and profile photographs may help elucidate such changes. MATERIAL AND METHODS: Photographs of 31 edentulous patients were compared in relaxed lip closure and after insertion of a CRDP in stable occlusion. 2232 anthropometric distances were raised. Eighteen anthropometric indices reflecting the perioral morphology and its integration in the vertical facial harmony were investigated. RESULTS: The intercanthal - mouth width index (p>.001), medial - lateral cutaneous upper lip height index (p=.007), lower vermilion contour index (p=.022), vermilion - total upper lip height index (p=.018), cutaneous - total upper lip height index (p=.023), upper lip - nose height index (p=.001), nose - upper face height index (p=.002), chin - mandible height index (p=.013), upper lip - mandible height index (p=.045), nose - lower face height index (p=.018), and nose - face height index (p=.029) showed significant pre- to post-treatment changes. CONCLUSIONS: The investigated anthropometric indices presented reproducible results related to an increase in occlusal vertical dimension. Their application may be helpful in assessment, planning, and explanation of morphologic effects of CRDPs on the perioral and overall facial morphology, which may helps to improve the aesthetic outcome.

Longterm quality of life after oncologic surgery and microvascular free flap reconstruction in patients with oral squamous cell carcinoma.

BACKGROUND: Quality of life (QoL) has become increasingly important in cancer treatment. It refers to the patient's perception of the effects of the disease and therapy, and their impact on daily functioning and general feeling of well being. MATERIAL AND METHODS: In this prospective study, a total of 100 patients treated at our institution, completed the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire and the specific EORTC QLQ-H&N35 module. The questionnaires were distributed to the patients between 12 and 60 months postoperatively. RESULTS: Global QoL score was 58.3 deterioration and mean score for functioning scale was 76.7. Fatigue (28.7 ± 26.1), followed by financial problems (27.7 ± 33.5), insomnia (26.7 ± 34.5) and pain (26.3 ± 29.9) had highest symptom score on QLQ-C30. Fatigue (r=-0.488), insomnia (r=-0.416) and pain (r =-0.448) showed highest value for significantly negative correlation to global QoL. In the H&N35 module, restriction of mouth opening (43.3 ± 38.6), dry mouth (40.7 ± 36.9), sticky saliva (37.3 ± 37.1) and eating in public (33.8 ± 31.9) were the four worst symptoms. Swallowing problem (r=-0.438), eating in public (r=-0.420) and persistent severe speech (r=-0.398) ranked as the three worst symptoms with highest value for significantly negative correlation to global QoL. CONCLUSIONS: Longterm QoL after oncologic surgery and microvascular free flap reconstruction in patients with oral cancer is satisfactory. Measurung QoL should be considered as part of the evaluation of cancer treatment.

VEGF transfer based on gene-modified fibroblasts using a hypoxia-induced vector to modulate neoangiogenesis in ischaemic regions of myocutaneous transplants.

The effect of a hypoxia-inducible VEGF-expressing on wound healing in an ischaemic hind leg rat model was evaluated in this study. 180 Wistar rats were assigned randomly to three groups. After ligation of the femoral artery, group 1 received pRTP801-VEGF165, group 2 untransfected fibroblasts, group 3 saline; injection was into the subcutaneous tissue, proximal and distal to the artery ligation. Biopsy specimens were obtained on days 3, 5, 7, 14 after implementation. VEGF transgene expression, vessel architecture, the amount and total area of vessel formation were investigated. Results showed a significantly higher level of VEGF protein expression in group 1 compared to group 2 (P≤0.001) throughout the investigational period. Group 1 exhibited a significant growth of CD31-positive blood vessels in the subcutaneous tissue on day 14 compared to groups 2 and 3 (P≤0.001) (group 1, 62.20±1.92; group 2, 20.60±1.67; group 3, 12.40±1.14). Alpha-SMA-positive staining also showed significant vessel growth in group 1 on day 5 (group 1, 27.00±1.87; group 2, 7.20±1.48; group 3, 10.00±1.73). These results were confirmed in the distal muscle tissue. No significant results were obtained for the proximal muscle tissue. The subcutaneous application of pRTP801-VEGF165 showed a long-lasting effect, with an increased expression of VEGF over the entire observation period. It appears that the use of fibroblasts transfected with VEGF is a promising way to increase early angiogenesis in ischaemic tissue.

Parotidectomy and submandibulectomy for benign diseases in Thuringia, Germany: a population-based study on epidemiology and outcome.

Although salivary gland surgery for benign diseases is an integral part of clinical routine of head and neck surgeons, there is not many population-based data published on incidence and efficiency of this surgery. Parotidectomy was performed in 180 patients and submandibulectomy in 97 patients for benign diseases in eight otorhinolaryngology and two maxillofacial surgery departments in Thuringia, Germany, in 2005. All patients were analysed regarding patients' characteristics, therapy, complications and further course of disease. Predominant indications were epithelial tumours for parotidectomy (79 %) and sialolithiasis for submandibulectomy (50 %). The most frequent tumour types were pleomorphic adenoma (46 %) and Warthin tumours (29 %). Pleomorphic adenoma was significantly more frequent in female patients and Warthin tumours in male patients and smokers. The incidence of parotidectomy, i.e. the surgical rate, was 7.8/100,000 habitants and of submandibulectomy 4.1/100,000 habitants. One hundred and seventy-eight tumours including 154 epithelial tumours resulted in an incidence of 7.6/100,000 habitants for all treated tumours and of 6.6/100,000 for epithelial tumours, respectively. The majority of parotid cases were treated by lateral parotidectomy (79 %). Relevant complications were observed in 22 % of patients. After parotidectomy and submandibulectomy a postoperative facial palsy was observed in 28 and 2 % of cases, respectively. Only 1 % was permanent. During a mean follow-up time of 9.6 months, 3 % of parotidectomy patients developed a Frey's syndrome needing treatment and 0.8 % developed a tumour recurrence. This population-based analysis shows that salivary gland surgery is performed in higher incidence than expected, effectively and with low-risk in daily routine of head and neck surgeons.

[The latissimus dorsi myocutaneous free flap--a salvage option for abdominal wall reconstruction in complex incisional hernias]. / Die M. latissimus dorsi-Lappenplastik--Salvage-Option bei der komplizierten Rezidiv-Narbenhernie.

Complex incisional hernias are a serious burden for the patient and a challenge for the surgeon. The main problem is the covering of the defect after the resection of large proportions of low-grade tissue. In this work, we describe 2 patients who underwent complex reconstruction of recurrent incisional hernias using a bovine pericardium mesh (Tutomesh®) and a latissimus dorsi myocutaneous free flap.

Lip reconstruction: an anthropometric and functional analysis of surgical outcomes.

Postoperative evaluation of results of reconstructive procedures of the lips mainly consists of describing the technique, complications and subjectively interpreted patient photographs. The authors present a photo-assisted pre- to postoperative evaluation of reconstructive procedures of the lips based on anthropometric measurements and functional outcome. Forty-eight patients underwent partial or full thickness excision of tumours of the lips and subsequent reconstruction. An analysis of standardized pre- and postoperative photographs included measurements of intercanthal width, mouth width, philtrum width, lateral upper lip height, upper and lower lip height, cutaneous upper and lower lip height, upper and lower vermilion arc and upper and lower vermilion height. Assessment of functional outcome consisted of the evaluation of oral competence, changes of oral opening and sensation. The effects on the described parameters were analysed, when partial or full thickness reconstruction had to be performed. Intercanthal mouth width index, vermilion arc index, vermilion and cutaneous total lower lip height index showed statistically significant effects postoperatively which indicates a postoperative tightened lower lip, when full-thickness excision was performed. In all patients the indices were proven reproducible and reliable. The standardized measurements described are accurate and objective for evaluating postoperative results.

Histomorphometric analysis of the phenotypical differentiation of recruited macrophages following subcutaneous implantation of an allogenous acellular dermal matrix.

This study aimed to clarify the phenotypical differentiation of recruited macrophages following subdermal implantation of an allogenous, acellular dermal matrix (aADM). In 20 male Wistar rats, one leg was randomly chosen for subcutaneous implantation of an aADM, while the other side received an autogenous dermis graft for control purposes. After 7 and 14 postoperative days, 10 animals were killed. Biopsies were obtained from the healing area and subjected to immunohistochemical staining (targets: pan macrophage marker CD68, M1 macrophage marker CD197, M2 macrophage marker CD163), histomorphometric analysis and reverse transcriptase polymerase chain reaction (targets: iNOS, arginase). No differences were detected in the total number of recruited macrophages between the groups. Allogenous ADMs significantly stimulated proinflammatory M1 differentiation, while autogenous dermis induced the regeneration promoting M2 phenotype. Proinflammatory M1 differentiation of macrophages might provide a potential explanation for profibrotic tissue deposition at the aADM interface following subcutaneous implantation, which has been observed previously.

Clinical-anthropometric and aesthetic analysis of nose and lip in unilateral cleft lip and palate patients.

OBJECTIVE: Presentation of a technique to determine objectively the degree of symmetry in the area of nose and lip in cleft patients based on analysis of photographs. To compare the objective measurements with the subjective impression. DESIGN: This was a retrospective study using a predefined photo documentation standard to capture images of the area of nose and lip. SETTING: Department of Oral and Maxillofacial Surgery, University of Jena, Germany. PATIENTS: Unilateral cleft patients following primary lip repair (group 1; n  =  36) or secondary correction (group 2; n  =  23). MAIN OUTCOME MEASURES: Measurements were taken on standardized photographs of three dimensions in the area of the nose and two dimensions in the upper lip region. Sign tests were used to ascertain differences between the cleft and unaffected sides separately for each group. Subjective impressions regarding symmetry were gathered and quantified by means of a visual analog scale (VAS). The Mann-Whitney U test was employed to compare the observers' impressions between the two groups. RESULTS: While significant side differences were found for all distances in group 1, only the side differences in the height of the nostril remained significant in group 2. Subjective evaluation of the nostril area improved significantly following corrective surgery. However, no such change in the area of the upper lip was recognized by the observers. CONCLUSIONS: We were able to demonstrate that the measurable symmetry of the nostril area, as well as the upper lip, was significantly enhanced by corrective surgery. However, only the subjective impression of the nostril was improved.

Head and neck in situ carcinoma: survival analysis of the Thuringian cancer registration database.

Head and neck in situ carcinoma is seldom diagnosed. Our knowledge about in situ cancer is limited. This study describes the epidemiology and prognosis of head and neck in situ cancer in Thuringia, Germany. We analyzed the cancer data of the Thuringian cancer registry database from 1996 to 2005. The database contained 3821 patients with primary head and neck cancer. Thirty-four patients (0.88%) had an in situ carcinoma. They were evaluated for patient's characteristics, tumor stage, incidence, treatment and trends in overall survival (OS) and recurrence-free survival (RFS). During 1996-2005, the average annual incidence of head and neck in situ carcinoma was 0.14 per 100,000 persons. Half of the cases were localized in the larynx. The patients were treated by local excision. Six patients (18%) developed a local recurrence. Only one recurrent tumor was diagnosed in early stage (rT1), but the other five tumors in advanced stage (rT3/rT4). The median time to recurrence was 27.43 months. For all 34 patients with in situ carcinoma, the 5-year OS was 84% and the 5-year RFS 60.4%. OS was better for laryngeal in situ cancer than for oral cavity or pharyngeal in situ cancer (p=0.031). The surveillance of patients with head and neck in situ carcinoma after treatment should be performed like in patients with invasive cancer, because nearly one fifth of patients developed a recurrence, predominantly in advanced stage.

Characterization of interfacial reactions between connective tissue and allogenous implants used for subdermal soft tissue augmentation.

The aim of this study was to investigate the molecular mechanisms underlying the problem of infection and pro-fibrotic tissue rearrangement around artificial devices used for soft tissue augmentation. An allogenous, acellularized dermal matrix (aADM) was implanted into subcutaneous pockets at the groin in 10 male Wistar rats. After 7 or 14 days, tissue specimens were obtained and analyzed for transforming growth factor (TGF)-beta1 and vascular endothelial growth factor (VEGF) by immunoblotting as well as alpha-smooth muscle actin (SMA)-and endoglin (CD105)-expression by immunohistochemistry. A TGFbeta1-dependent alpha-SMA-overexpression as well as capillary lumina area regression were observed in the peri-implant connective tissue. VEGF expression decreased over the entire investigation period and resulted in delayed neovascularization of the implant. In conclusion, these experiments demonstrate that co-delivery of anti-fibrotic and pro-angiogenic agents is highly desirable to improve the clinical success of artificial tissue substitutes.

Free flap transfer in cranio-maxillofacial surgery: a review of the current data.

BACKGROUND: The advances of cranio-maxillofacilal surgery are considerably driven by the evolution of microsurgical techniques. At present, these methods continue to provide new therapeutic options to the field. Especially, free flap transfer has evolved to become an integral part of current treatment protocols for head and neck malignancies. It ensures uneventful wound healing even after previous radiotherapy and can often preserve form and function. For many patients, this may lead to a significant improvement in their quality of life. OBJECTIVES: This review summarizes aspects of tumor therapy, the impact of radiation, and discusses different techniques of microvascular tissue transfer. DISCUSSION: Specific advantages in different anatomical sites of the head and neck region are highlighted in contrast to existing alternatives. Selected cases exemplify the use of popular transplants. SUMMARY: While planning reconstructions, it is important to consider both the functional and aesthetic aspects. The best individual outcome is based on a thoughtful match of available methods to a given defect and the patient's condition.

Evaluation of substitutes for bone: comparison of microradiographic and histological assessments.

We created defects of standard size in the frontal bones of adult pigs and filled them with four different materials. On six occasions (at 1, 2, 4, 8, 12, and 26 weeks), samples were harvested, and evaluated by computing microradiographic images. We examined the specimens histologically as controls. After insertion of anorganic materials, microradiographic evaluation was easy and precise, and there were no significant differences between them and the histological controls (p=0.2). A quantitative evaluation of chemically sterilised bone by computer was not possible for more than 4 weeks.

Changes in neurophysiologic parameters in a patient with dental anxiety by hypnosis during surgical treatment.

It was hypothesized that dental anxiety, which leads to neurophysiologic alterations in heart rate, respiratory rate and blood pressure prior, during and subsequent to dental treatment, can be influenced by medical hypnosis. We report the positive impact from non-invasive hypno-sedation during dental implant surgery on a 54-year-old female patient who experienced neurophysiologic reactions as a result of the psychosomatic process of dental anxiety (dental anxiety scale value = 13). The neurophysiologic changes during dental surgery performed with and without hypnosis were compared after the patient underwent the same surgical treatment protocol. This case report was part of a study designed to evaluate hypnosis as a non-invasive therapy for dental-anxious patients over six sessions using subjective experience and objective parameters, which included electroencephalogram, electrocardiogram, heart rate, blood pressure, oxygen saturation of the blood, respiration rate, salivary cortisol concentration and body temperature.

Expression of interleukin 1-beta, transforming growth factor beta-1, and vascular endothelial growth factor in soft tissue over the implant before uncovering.

BACKGROUND: Overexpression of inflammatory cytokines interleukin 1-beta (IL-1beta), transforming growth factor beta-1 (TGF-beta1), and vascular endothelial growth factor (VEGF) may cause healing impairment following implant insertion, jeopardizing success especially in patients previously irradiated. Limited data is available regarding expression pattern of inflammatory cytokines in peri-implant soft tissue caused by the surgical intervention itself. STUDY DESIGN: This study examined 21 patients receiving dental implants. Biopsies of peri-implant tissue were harvested at re-entry 4 months after initial surgery. Eight patients underwent probing of untreated mucosa. Three groups were created (group 1: regular peri-implant mucosa; group 2: patients with irradiated peri-implant mucosa, radiation treatment due to oral squamous cell cancer; group 3: control). Immunohistochemical staining was performed for TGFss1, IL-1ss, and VEGF. RESULTS: Following the placement of dental implants (group 1 vs group 3) a significant increase (P > .05) in TGF-beta1, IL-1beta, and VEGF expression in the peri-implant mucosa was demonstrated. No alteration of this distinct pattern was found for previously irradiated tissue (group 1 vs. group 2). CONCLUSIONS: The study highlights the fundamental involvement of TGF-beta1, IL-1beta, and VEGF during the regeneration of peri-implant soft tissue structures. The use of extended interim solutions may be one clinical implication of these prolonged tissue remodeling processes.

Onlay augmentation versus sinuslift procedure in the treatment of the severely resorbed maxilla: a 5-year comparative longitudinal study.

Augmentation procedures in the severely resorbed maxilla are standard techniques in preprosthetic surgery. Aim of the present study was to compare onlay bone grafting and sinus floor elevation with autogenous bone grafts in edentulous patients in terms of bone resorption of the posterior alveolar crest and implant survival in a 5-year longitudinal study. A total of 100 patients (53 female and 47 male patients) requiring augmentation using autogenous bone due to a severely resorbed maxillae were evaluated. The degree of resorption and bone quality were preoperatively estimated in panoramic radiographs according to Lekholm and Zarb. The overall 5-year success rate of implants placed after augmentation procedures in the posterior maxilla was 93.1%. Following onlay augmentations 215 out of 235 implant insertions were successful, i.e. a success rate of 91.5% was achieved. In the sinuslift group, 330 out 349 implants remained successful, i.e. a success rate of 94.6% was found. The difference between both groups was statistically significant at the end of the evaluation period. Bone resorption, measured as loss of total bone height in panoramic radiographs, was initially more pronounced following onlay augmentations (>20% after 12 months of observation). The degree of resorption decreased in the further course of the study. Lower resorption rates and a higher overall success rate were demonstrated for the sinus lift group.

[Patients with oral squamous cell carcinoma. Long-term survival and evaluation of quality of life-initial results obtained with two treatment protocols in a prospective study]. / Plattenepithelkarzinome der Mundhöhle. Uberlebenswahrscheinlichkeit und Lebensqualität-Erste Ergebnisse von 2 Behandlungsstrategien.

INTRODUCTION: In patients with oral cancer the treatment has a strong impact on the quality of life. In recent years different therapeutic concepts have been developed, which include preoperative simultaneous 'neoadjuvant' radiochemotherapy (RCT) and one-stage surgery with tumour ablation and reconstruction. Consideration of long-term survival rates yields substantial evidence that mixed-modality treatment including neoadjuvant RCT is superior to adjuvant therapy concepts based on a surgical approach with postoperative radiation. PATIENTS AND METHODS: In this nonrandomised longitudinal prospective study quality of life was evaluated in two groups made up of a total of 53 patients with squamous cell carcinoma of the oral cavity, 26 of whom underwent neoadjuvant radiochemotherapy with subsequent surgical resection while the remaining 27 received surgical treatment first and then postoperative radiotherapy. The quality-of-life core questionnaire (QLQ C-30) and the head and neck cancer module (H&N 35) of the European Organisation for Research and Treatment of Cancer (EORTC) were used. Long-term survival was estimated according to the Kaplan-Meier test. RESULTS: Postoperatively both groups showed a marked reduction in quality of life, especially in restricted chewing, swallowing and speaking. One year later their quality of life had improved substantially, though without quite reaching the preoperative quality-of-life scores. Both groups showed specific impairments in the symptom scales. With adjustment for the fact that the patients were not randomised, long-term survival was 78% in the neoadjuvant treatment group and 50% in the adjuvant treatment group. CONCLUSION: Temporary limitations in the quality of life can be expected after tumour treatment of the kinds presented here for oral cancer. Neoadjuvant therapy concept is more aggressive and might result in longer disease-free survival. The primary goal should be eradication of the tumour. Nevertheless preservation or reconstruction of a maximum of function is essential for a high level of quality of life. Combined-modality treatments seem to be superior to any kind of monotherapy and should therefore be preferred.

In vitro cultured autologous pre-confluent oral keratinocytes for experimental prefabrication of oral mucosa.

The reconstruction of large defects after head and neck cancer resection often requires composite tissue transfer to replace a combination of bone, muscle and mucosa. Thus, tissue engineering techniques may be useful for oral mucosal reconstructive surgery to prefabricate mucosal tissue on the muscle flap in vivo, instead of using conventional skin-bearing composite flaps. The aim of this study was to investigate whether autogenous pre-confluent oral keratinocytes (PCOK) cultured in vitro can create mucosal coverage on muscle in vivo, in a single grafting procedure. In 30 Wistar rats, with a small piece of oral mucosa (2 mm x 5 mm), oral keratinocytes were isolated and then seeded on a hydrophilic PTFE membrane (n = 50) in serum-free culture condition. After 48 h, the membrane, together with the PCOK, was transplanted onto the gracilis muscle to fabricate a mucosal flap in vivo. The wound bed was closed primarily until the time of examination. Biopsies were carried out 1, 2, 3, and 4 weeks, respectively, after transplantation and were evaluated immunohistochemically (AE1/AE3 anti-pancytokeratin, cytokeratin 5/6, collagen IV, laminin, lectin-specific labeling of N-acetylglucosamine oligomeres of endothelial cells) with relation to the following criteria: (1) graft acceptance; (2) inflammatory signs; (3) structural changes and keratinocyte lining; (4) expression of basement membrane components; and (5) vascularization. Ninety-one percent of the grafts showed uniform epithelial layers. The mean number of reconstructed epithelial cell layers was 1.7, 2.0, 1.85 and 2.7 at 1, 2, 3 and 4 weeks, respectively after transplantation (P = 0.342). Collagen IV, laminin and lectin-specific capillaries developed between the neoepithelium and the underlying muscular layer. Only two specimens showed signs of infection 2 weeks after transplantation. In conclusion, this experiment demonstrated that PCOK grafts on muscle in vivo can achieve uniform multi-layered oral epithelial coverage in a short period of time. This technique may be a useful alternative tool for oropharyngeal reconstructive surgery and is also worth considering for further clinical studies.